Cytology , Diagnostics

Cytology of Neoplasia: An Essential Component of Diagnosis

Cytology of Neoplasia: An Essential Component of Diagnosis
  •  
  •  
  •  
  •  
  •  
  •  
  •  

pdf_button


Anne Barger, DVM, MS, Diplomate ACVP

Cytology is a quick, easy, and inexpensive diagnostic tool. It is commonly used for the diagnosis of neoplastic processes.

Cytology has several benefits:

  • The risks of fine-needle aspiration are minimal
  • Anesthesia or sedation is often not required
  • Aspirated tissue does not require any processing—results can be obtained more quickly than with histopathology.

However, cytology is not without limitations: due to lack of tissue architecture and variability of cellularity, cytologic diagnoses can be misinterpreted or inconclusive. Cytology is often used in conjunction with biopsy or additional cytochemical or immunocytochemical stains to improve specificity of diagnosis.

Cytologic Classification

Many neoplasms present as discrete masses in the skin or subcutaneous tissue or within an organ. It is important to understand the cytology of healthy tissue cells in order to recognize a pathologic process, such as a tumor.

Classification is based on cellular morphology and tissue of origin, if recognizable. Cytologically, neoplasms are classified into 1 of 3 categories, with rare exceptions:

  1. Epithelial neoplasia
  2. Mesenchymal neoplasia
  3. Round cell neoplasia.

Benign neoplasms should consist of a uniform population of cells; however, it is essential that specimens be thoroughly evaluated for criteria of malignancy, which include:

  • Anisocytosis
  • Anisokaryosis
  • Prominent/multiple nucleoli
  • Variation in the nucleus to cytoplasmic ratio (N:C)
  • Multiple nuclei
  • Abnormal mitotic figures.

What About Melanoma?

Some text books classify melanomas as round cell tumors. However, the cells can exhibit features similar to epithelial, mesenchymal, and round cell tumors, occasionally in the same tumor, which is why they can be difficult to characterize.

Figure A

Figure A. Benign melanoma from a cat: The cells contain so many granules that individual cellular features are difficult to discern. (Wright-Giemsa stain; 1000x)

Benign melanomas generally contain so much pigment within the cytoplasm that it is difficult to fully evaluate the cellular detail.

Figure B

Figure B. Malignant melanoma from a dog: The cells exhibit obvious criteria of malignancy, including large prominent nucleoli, and have minimal granulation in the cytoplasm. (Wright-Giemsa stain; 1000x)

Malignant melanomas occur most commonly in the oral cavity or nail bed. They often have much less pigment than benign melanomas, making criteria of malignancy, such as a single large prominent nucleoli, easy to spot.

Epithelial Neoplasia

Appearance

Epithelial cells commonly are cohesive; therefore, cytologically, epithelial tumors are arranged in tightly adhered clusters. The cells are often round with round nuclei, though some epithelial neoplasms, such as squamous cell carcinomas, can have atypical shapes.

N:C varies greatly depending on cell type and tissue of origin:

  • Epithelial cells from healthy tissue (liver, pancreas, lung, etc) are uniform in size and shape with distinct cell borders.
  • Neoplastic epithelial cells from glandular tissue may have cytoplasmic vacuoles or produce a cytoplasmic product, appearing as discrete granules or as a large globule that displaces the nucleus.
  • Carcinomas will generally have marked criteria of malignancy and, in the absence of an inflammatory process, can be easy to distinguish from healthy tissue.

Types of Tumors

Some of the more common cutaneous epithelial tumors include:

  • Sebaceous adenoma
  • Trichoblastoma
  • Squamous cell carcinoma.

Sebaceous adenomas are benign and fairly common in dogs but less so in cats.1 Surgical excision should be curative. See Table 1 for a list of cytologic characteristics.

Table 1. Cytologic Characteristics of Epithelial Neoplasia
Sebaceous Adenomas
  • Clusters of uniform glandular epithelial cells
  • Cytoplasm: Abundant and filled with discrete cytoplasmic vacuoles
  • Nucleus: Often obscured by vacuoles
  • Difficult to differentiate from sebaceous hyperplasia due to lack of tissue architecture
Basal Cell Tumors
  • Small, round cells that occur in clusters or linear rows or ribbons
  • Cytoplasm: Scant rim of pale basophilic cytoplasm
  • Nucleus: Large and round, with clumped chromatin pattern
  • Have high nucleus:cytoplasmic ratio
Trichoblastomas
  • Cells are often larger than those of basal cell tumors
  • Cytoplasm: Scant rim of basophilic cytoplasm
  • Nuclei: Prominent, large and round
Squamous Cell Carcinomas
  • Round to polygonal shaped cells
  • Cytoplasm: Basophilic in varying amounts; often keratinized
  • Nuclei: Round; vary greatly in size and chromatin pattern, from coarsely stippled to pyknotic; perinuclear vacuoles common
  • Often marked variation in nucleus:cytoplasmic ratio because cells vary greatly in level of differentiation

Trichoblastomas (Figure 1) in dogs were previously known as benign basal cell tumors; they were reclassified as trichoblastomas because, histopathologically, the tumor shows differentiation of the hair germ of the developing follicle.2 They occur most commonly around the head and neck and occasionally metastasize. See Table 1 for a list of cytologic characteristics.3

f01_fig1-2

Squamous cell carcinomas (Figure 2) are the most common head and neck tumor,4 one of the most common epithelial tumors of the nose and paranasal sinus in cats,5 and a fairly common oral and nasal tumor in dogs. Cytologic diagnosis of squamous cell carcinoma can be straightforward; see Table 1 for a list of cytologic characteristics. These tumors are often ulcerated and necrotic so secondary suppurative inflammation is common.

Mesenchymal Neoplasia

Appearance
Mesenchymal tumors originate from connective tissue. Connective tissue includes fibrous, adipose, bone, cartilage, and muscle connective tissue.

  • Mesenchymal cells can vary in shape, from round to spindle, and have wispy cytoplasmic borders.
  • The nuclei are often oval but occasionally round.

Healthy mesenchymal tissue does not exfoliate well but malignant mesenchymal tissue exfoliates readily; cellularity can assist with a diagnosis of neoplasia.

Types of Tumors

  • Sarcomas, such as osteosarcoma and hemangiosarcoma, exhibit obvious criteria of malignancy.
  • However, other mesenchymal tumors (classified as soft tissue sarcomas on histopathology) display minimal criteria for malignancy.
  • This histopathologic subheading includes a number of tumors, including:
    • Perivascular wall tumors, such as myopericytomas and hemangiopericytomas
    • Peripheral nerve sheath tumors, such as neurofibromas and schwannomas.
Table 2. Cytologic Characteristics of Mesenchymal Neoplasia
Osteosarcomas
  • Majority consist of plasmacytoid cells
  • Cytoplasm: Basophilic in varying amounts; often contains eosinophilic granules (common to find eosinophilic product within background; ie, osteoid)
  • Nuclei: Large, prominent; often eccentrically placed in cell
  • Often marked anisocytosis and anisokaryosis plus multinucleation
  • Erythrophagia occasionally observed in neoplastic osteoblasts;1 low numbers of osteoblasts observed in these specimens
Hemangiosarcomas
  • Spindle-shaped, plump cells
  • Nuclei: Large, prominent
  • Moderate anisocytosis and anisokaryosis
  • Erythrophagia occasionally observed
Soft Tissue Sarcomas
  • Unusually shaped cells
  • Cytoplasm: Very long, often bipolar, projections; can contain low numbers of discrete vacuoles
  • Nuclei: Prominent and round, with a stippled chromatin pattern; multinucleated cells containing 5 to 6 nuclei are common
  • Red blood cells often identified in background
1. Barger A, Skowronski M, MacNeill A. Cytologic identification of erythrophagocytic neoplasms in dogs. Accepted for publication; Vet Clin Pathol.

Osteosarcomas (Figure 3) can be difficult to differentiate from other primary bone tumors. A cytochemical stain is available to help differentiate osteosarcoma from other mesenchymal tumors. One limitation of this stain is that normal or reactive osteoblasts will also stain positively; therefore, close examination of the aspirate for clear evidence of malignancy is recommended before this stain is performed.6 See Table 2 for a list of cytologic characteristics.

Hemangiosarcomas (Figure 4) can occur in multiple locations, including the spleen, liver, bone, skin, muscle, and heart. Thoracic and abdominal hemangiosarcomas are often accompanied by significant hemorrhage. Masses are often cavitated, making it difficult to obtain a diagnostic sample.

If possible, aspiration of a more solid portion of the mass is beneficial. See Table 2 for a list of cytologic characteristics. Histopathologically, hemangiosarcomas are occasionally described as epithelioid and have solid areas, which result in a more cellular aspirate.7

Soft tissue sarcoma (Figure 5) aspirates are often cellular and consist of aggregates and individualized mesenchymal cells. See Table 2 for a list of cytologic characteristics. However, these sarcomas cannot be classified as malignant based on cytology. Instead, histopathologic grading and immunohistochemistry are recommended to predict the behavior of these tumors.8

Lipomas (Figure 6) are benign tumors composed of mature adipocytes. Cytologically, a lipoma cannot be distinguished from subcutaneous adipose tissue. Grossly, the aspirate itself will consist of droplets of lipid that often rinse away during the staining process. Intact adipocytes are large cells with voluminous amounts of cytoplasm and small round nuclei.

f01_fig3,4,5,6

Round Cell Neoplasia

Appearance
These tumors have many similar features that include:

  • Round cell shape
  • Arrangement of cells individually or in sheets.

These tumors exfoliate very well, resulting in highly cellular cytologic specimens. Each round cell tumor has distinct features.

Types of Tumors
Round cell tumors consist of several different tumor types; the most common include:

  • Plasma cell tumors
  • Histiocytomas
  • Mast cell tumors
  • Lymphoma
  • Transmissible venereal tumors.

Plasma cell tumors (Figure 7) can be difficult to distinguish from histiocytoma, especially if a prominent golgi apparatus is not visible. See Table 3 for a list of cytologic characteristics.

Histiocytomas (Figure 8) are only identified in dogs and commonly diagnosed in dogs less than 4 years of age.1 The neoplastic cell is a Langerhans cell or epidermal dendritic cell9 and often resolves on its own. See Table 3 for a list of cytologic characteristics.

Mast cell tumors (Figure 9) are the most common skin tumor in dogs.10 They can occur as single or multiple skin tumors and occasionally infiltrate the abdominal organs and bone marrow. Mast cell tumors cannot be graded on cytology; histopathology is required. Aspiration of the draining lymph nodes may be beneficial to determine the potential behavior of the tumor. See Table 3, page 16, for a list of cytologic characteristics.

Mast cell granules stain consistently with Giemsa, new methylene blue, and toluidine blue stains; however, they do not consistently stain with aqueous-based Romanowsky stains, such as Diff-Quik. When doing in-house cytology, it is important to leave at least 1 slide unstained; then if the slide is sent to a laboratory, the stains mentioned previously can be performed.

f01_fig7,8,9

Lymphomas (Figure 10) are more commonly a pure population of large, intermediate or, less likely, small lymphocytes. They appear cytologically similar whether in lymph nodes, skin, or visceral organs. Biopsy with histopathology, polymerase chain reaction for T- and B-cell gene rearrangements, and immunocytochemical staining can be beneficial when characterizing or classifying this neoplasm. See Table 3 for a list of cytologic characteristics.

Transmissible venereal tumors (Figure 11) are found in dogs, wolves, and coyotes and transmitted primarily by physical contact.11 See Table 3 for a list of cytologic characteristics.

f01_fig10-11

Table 3. Cytologic Characteristics of Round Cell Neoplasia
Plasma Cell Tumors
  • Cytoplasm: Basophilic in moderate amount
  • Nucleus: Eccentrically placed; binucleated and multinucleated cells commonly observed
  • Often a perinuclear clearing or prominent golgi apparatus observed
Histiocytomas
  • Sheets of uniform round cells
  • Cytoplasm: Moderate rim of pale basophilic cytoplasm; distinguishing characteristic is clearing of cytoplasm at periphery of cell
  • Nucleus: Often centrally placed
  • As tumors begin to resolve, increasing numbers of small lymphocytes are seen
Mast Cell Tumors
  • Cytoplasm: Basophilic in moderate amount, containing varying numbers of metachromatic granules
  • Nucleus: Round and centrally placed; often chromatin pattern and nucleoli cannot be fully evaluated due to intense granulation
  • Eosinophils often seen in dogs; less frequently in cats
Lymphoma
  • Round cells
  • Cytoplasm: Scant rim of basophilic cytoplasm; occasionally has low numbers of eosinophilic granules
  • Nucleus: Large and round; lymphoma consisting of large- or intermediate-sized lymphocytes likely has prominent nuclei
Transmissible Venereal Tumors
  • Chromatin pattern is coarse with occasional binucleation
  • Moderate amount of pale basophilic cytoplasm; often contains several small discrete cytoplasmic vacuoles

In Summary

Cytology is a useful diagnostic tool, especially in the evaluation of neoplasia. It is a noninvasive technique with sometimes immediate results. Identification of a malignant process can be done easily in many cases and may assist veterinarians in planning the next diagnostic step.

N:C = nucleus to cytoplasmic ratio

References

  1. Goldschmidt MH, Hendrick MJ. Tumors of the skin and soft tissues. In Meuten DJ (ed): Tumors in Domestic Animals, 4th ed. Ames, IA: Iowa State Press, 2002.
  2. Sharif M, Reinacher M. Clear cell trichoblastomas in two dogs. J Vet Med 2006; 53:352-354.
  3. Stockhaus C, Teske E. Assessment of cytological criteria for diagnosing basal cell tumours in the dog and cat. J Small Anim Pract 2001; 42:582-586.
  4. Fidel J, Lyons J, Tripp C, et al. Treatment of oral squamous cell carcinoma with accelerated radiation therapy and concomitant carboplatin in cats. J Vet Int Med 2011; 25(3):504-510.
  5. Mukaratirwa S, van der Linde-Sipman JS, Gruys E. Feline nasal and paranasal sinus tumours: Clinicopathological study, histomorphological description and diagnostic immunohistochemistry of 123 cases. J Fel Med Surg 2001; 3:235-245.
  6. Barger A, Graca R, Bailey K, et al. Use of alkaline phosphatase staining to differentiate canine osteosarcoma from other vimentin positive tumors. Vet Pathol 2005; 42:161-165.
  7. Shor S, Helfand SC, Gorman E, Löhr CV. Diagnostic exercise: Epithelioid hemangiosarcoma mimicking metastatic prostatic neoplasia in a dog. Vet Pathol 2009; 46:548-552.
  8. Avallone G, Helmbold P, Caniatti M, et al. The spectrum of canine cutaneous perivascular wall tumors: Morphologic, phenotypic and clinical characterization. Vet Pathol 2007; 44:607-620.
  9. Affolter KV, Moore PF. Canine cutaneous systemic histiocytosis: Reactive histiocytosis of dermal dendritic cells. Am J Dermatopath 2000; 22:40-48.
  10. London CA, Seguin B. Mast cell tumors in the dog. Vet Clin North Am Small Anim 2003; 33:473-489.
  11. von Holdt BM, Ostrander EA. The singular history of a canine transmissible tumor. Cell 2006; 126:445-447.

f01_BargerAnne Barger, DVM, MS, Diplomate ACVP, is a clinical associate professor in pathobiology at the University of Illinois College of Veterinary Medicine and the section head of clinical pathology for its Veterinary Diagnostic Laboratory. Her research interests include immunocytochemistry, osteosarcoma, and cytology, and she is a prolific writer on these topics. Dr. Barger is a member of the American College of Veterinary Pathology’s exam committee and the American Society for Veterinary Clinical Pathology’s membership committee. She received her DVM and MS from University of Illinois and completed a residency in clinical pathology at North Carolina State University.

DMCA.com Protection Status
[gravityform id="6" title="false" description="false"]
<div class='gf_browser_gecko gform_wrapper' id='gform_wrapper_6' style='display:none'><form method='post' enctype='multipart/form-data' id='gform_6' action='/cytology-of-neoplasia-an-essential-component-of-diagnosis/'> <div class='gform_body'><ul id='gform_fields_6' class='gform_fields top_label form_sublabel_below description_below'><li id='field_6_1' class='gfield gfield_contains_required field_sublabel_below field_description_below gfield_visibility_visible' ><label class='gfield_label gfield_label_before_complex' >Name<span class='gfield_required'>*</span></label><div class='ginput_complex ginput_container no_prefix has_first_name no_middle_name has_last_name no_suffix gf_name_has_2 ginput_container_name' id='input_6_1'> <span id='input_6_1_3_container' class='name_first' > <input type='text' name='input_1.3' id='input_6_1_3' value='' aria-label='First name' aria-required="true" aria-invalid="false" /> <label for='input_6_1_3' >First</label> </span> <span id='input_6_1_6_container' class='name_last' > <input type='text' name='input_1.6' id='input_6_1_6' value='' aria-label='Last name' aria-required="true" aria-invalid="false" /> <label for='input_6_1_6' >Last</label> </span> </div></li><li id='field_6_2' class='gfield gfield_contains_required field_sublabel_below field_description_below gfield_visibility_visible' ><label class='gfield_label' for='input_6_2' >Email<span class='gfield_required'>*</span></label><div class='ginput_container ginput_container_email'> <input name='input_2' id='input_6_2' type='text' value='' class='medium' aria-required="true" aria-invalid="false" /> </div></li><li id='field_6_7' class='gfield gfield_contains_required field_sublabel_below field_description_below gfield_visibility_visible' ><label class='gfield_label' for='input_6_7' >Profession / Role<span class='gfield_required'>*</span></label><div class='ginput_container ginput_container_select'><select name='input_7' id='input_6_7' class='medium gfield_select' aria-required="true" aria-invalid="false"><option value='Veterinarian' >Veterinarian</option><option value='Veterinary Nurse / Technician' >Veterinary Nurse / Technician</option><option value='Practice Manager / Veterinary Assistant / Support Staff' >Practice Manager / Veterinary Assistant / Support Staff</option><option value='Student (Veterinarian or Veterinary Nurse)' >Student (Veterinarian or Veterinary Nurse)</option><option value='Other' >Other</option></select></div></li><li id='field_6_20' class='gfield gfield_html gfield_html_formatted gfield_no_follows_desc field_sublabel_below field_description_below gfield_visibility_visible' ><br></li><li id='field_6_15' class='gfield field_sublabel_below field_description_above gfield_visibility_visible' ><label class='gfield_label' >Inbox Preferences</label><div class='gfield_description' id='gfield_description_6_15'>Customize your inbox by selecting the areas you are most interested in. </div><div class='ginput_container ginput_container_checkbox'><ul class='gfield_checkbox' id='input_6_15'><li class='gchoice_6_15_1'> <input name='input_15.1' type='checkbox' value='NAVC Better Care Weekly&lt;br&gt;&lt;div style=&quot;font-size: 12px;color: #808080&quot;&gt;A weekly roundup of the new and noteworthy innovations and educational opportunities in the veterinary industry.&lt;/div&gt;' checked='checked' id='choice_6_15_1' /> <label for='choice_6_15_1' id='label_6_15_1'>NAVC Better Care Weekly<br><div style="font-size: 12px; color: #808080;">A weekly roundup of the new and noteworthy innovations and educational opportunities in the veterinary industry.</div></label> </li><li class='gchoice_6_15_2'> <input name='input_15.2' type='checkbox' value='Today&#039;s Veterinary Business eNewsletter&lt;br&gt;&lt;div style=&quot;font-size: 12px;color: #808080&quot;&gt;Covers topics relating to practice management, financial metrics, marketing, retailing and merchandising, and industry news.&lt;/div&gt;' id='choice_6_15_2' /> <label for='choice_6_15_2' id='label_6_15_2'>Today's Veterinary Business eNewsletter<br><div style="font-size: 12px; color: #808080;">Covers topics relating to practice management, financial metrics, marketing, retailing and merchandising, and industry news.</div></label> </li><li class='gchoice_6_15_3'> <input name='input_15.3' type='checkbox' value='Today&#039;s Veterinary Nurse eNewsletter&lt;br&gt;&lt;div style=&quot;font-size: 12px;color: #808080&quot;&gt;Provides practical, peer-reviewed companion animal educational CE for veterinary nurses, technicians and assistants.&lt;/div&gt;' id='choice_6_15_3' /> <label for='choice_6_15_3' id='label_6_15_3'>Today's Veterinary Nurse eNewsletter<br><div style="font-size: 12px; color: #808080;">Provides practical, peer-reviewed companion animal educational CE for veterinary nurses, technicians and assistants.</div></label> </li><li class='gchoice_6_15_4'> <input name='input_15.4' type='checkbox' value='Today&#039;s Veterinary Practice eNewsletter&lt;br&gt;&lt;div style=&quot;font-size: 12px;color: #808080&quot;&gt;Provides practical, peer-reviewed articles focused on the latest diagnostic and treatment techniques to enhance your knowledge and confidence.&lt;/div&gt;' id='choice_6_15_4' /> <label for='choice_6_15_4' id='label_6_15_4'>Today's Veterinary Practice eNewsletter<br><div style="font-size: 12px; color: #808080;">Provides practical, peer-reviewed articles focused on the latest diagnostic and treatment techniques to enhance your knowledge and confidence.</div></label> </li><li class='gchoice_6_15_5'> <input name='input_15.5' type='checkbox' value='VetFolio eNewsletter&lt;br&gt;&lt;div style=&quot;font-size: 12px;color: #808080&quot;&gt;Discover online CE resources offered in a variety of formats including videos, podcasts, webinars and more.&lt;/div&gt;' id='choice_6_15_5' /> <label for='choice_6_15_5' id='label_6_15_5'>VetFolio eNewsletter<br><div style="font-size: 12px; color: #808080;">Discover online CE resources offered in a variety of formats including videos, podcasts, webinars and more.</div></label> </li><li class='gchoice_6_15_6'> <input name='input_15.6' type='checkbox' value='Partner Emails&lt;br&gt;&lt;div style=&quot;font-size: 12px;color: #808080&quot;&gt;Exclusive deals and information from our partners.&lt;/div&gt;' id='choice_6_15_6' /> <label for='choice_6_15_6' id='label_6_15_6'>Partner Emails<br><div style="font-size: 12px; color: #808080;">Exclusive deals and information from our partners.</div></label> </li></ul></div></li><li id='field_6_19' class='gfield gfield_html gfield_html_formatted field_sublabel_below field_description_below gfield_visibility_visible' ><br></li><li id='field_6_22' class='gfield field_sublabel_below field_description_below gfield_visibility_visible' ><label class='gfield_label' >Do you want to be added to our postal mailing list?</label><div class='ginput_container ginput_container_checkbox'><ul class='gfield_checkbox' id='input_6_22'><li class='gchoice_6_22_1'> <input name='input_22.1' type='checkbox' value='Yes, I love mail!' id='choice_6_22_1' /> <label for='choice_6_22_1' id='label_6_22_1'>Yes, I love mail!</label> </li><li class='gchoice_6_22_2'> <input name='input_22.2' type='checkbox' value='No thank you.' id='choice_6_22_2' /> <label for='choice_6_22_2' id='label_6_22_2'>No thank you.</label> </li></ul></div></li><li id='field_6_10' class='gfield gfield_contains_required field_sublabel_below field_description_below gfield_visibility_visible' ><label class='gfield_label gfield_label_before_complex' >Please provide your mailing address below.<span class='gfield_required'>*</span></label> <div class='ginput_complex ginput_container has_street has_street2 has_city has_state has_zip has_country ginput_container_address' id='input_6_10' > <span class='ginput_full address_line_1' id='input_6_10_1_container' > <input type='text' name='input_10.1' id='input_6_10_1' value='' aria-required="true"/> <label for='input_6_10_1' id='input_6_10_1_label' >Street Address</label> </span><span class='ginput_full address_line_2' id='input_6_10_2_container' > <input type='text' name='input_10.2' id='input_6_10_2' value='' /> <label for='input_6_10_2' id='input_6_10_2_label' >Address Line 2</label> </span><span class='ginput_left address_city' id='input_6_10_3_container' > <input type='text' name='input_10.3' id='input_6_10_3' value='' aria-required="true"/> <label for='input_6_10_3' id='input_6_10_3_label' >City</label> </span><span class='ginput_right address_state' id='input_6_10_4_container' > <input type='text' name='input_10.4' id='input_6_10_4' value='' aria-required="true"/> <label for='input_6_10_4' id='input_6_10_4_label' >State / Province / Region</label> </span><span class='ginput_left address_zip' id='input_6_10_5_container' > <input type='text' name='input_10.5' id='input_6_10_5' value='' aria-required="true"/> <label for='input_6_10_5' id='input_6_10_5_label' >ZIP / Postal Code</label> </span><span class='ginput_right address_country' id='input_6_10_6_container' > <select name='input_10.6' id='input_6_10_6' aria-required="true"><option value='' selected='selected'></option><option value='Afghanistan' >Afghanistan</option><option value='Åland Islands' >Åland Islands</option><option value='Albania' >Albania</option><option value='Algeria' >Algeria</option><option value='American Samoa' >American Samoa</option><option value='Andorra' >Andorra</option><option value='Angola' >Angola</option><option value='Anguilla' >Anguilla</option><option value='Antarctica' >Antarctica</option><option value='Antigua and Barbuda' >Antigua and Barbuda</option><option value='Argentina' >Argentina</option><option value='Armenia' >Armenia</option><option value='Aruba' >Aruba</option><option value='Australia' >Australia</option><option value='Austria' >Austria</option><option value='Azerbaijan' >Azerbaijan</option><option value='Bahamas' >Bahamas</option><option value='Bahrain' >Bahrain</option><option value='Bangladesh' >Bangladesh</option><option value='Barbados' >Barbados</option><option value='Belarus' >Belarus</option><option value='Belgium' >Belgium</option><option value='Belize' >Belize</option><option value='Benin' >Benin</option><option value='Bermuda' >Bermuda</option><option value='Bhutan' >Bhutan</option><option value='Bolivia' >Bolivia</option><option value='Bonaire, Sint Eustatius and Saba' >Bonaire, Sint Eustatius and Saba</option><option value='Bosnia and Herzegovina' >Bosnia and Herzegovina</option><option value='Botswana' >Botswana</option><option value='Bouvet Island' >Bouvet Island</option><option value='Brazil' >Brazil</option><option value='British Indian Ocean Territory' >British Indian Ocean Territory</option><option value='Brunei Darussalam' >Brunei Darussalam</option><option value='Bulgaria' >Bulgaria</option><option value='Burkina Faso' >Burkina Faso</option><option value='Burundi' >Burundi</option><option value='Cambodia' >Cambodia</option><option value='Cameroon' >Cameroon</option><option value='Canada' >Canada</option><option value='Cape Verde' >Cape Verde</option><option value='Cayman Islands' >Cayman Islands</option><option value='Central African Republic' >Central African Republic</option><option value='Chad' >Chad</option><option value='Chile' >Chile</option><option value='China' >China</option><option value='Christmas Island' >Christmas Island</option><option value='Cocos Islands' >Cocos Islands</option><option value='Colombia' >Colombia</option><option value='Comoros' >Comoros</option><option value='Congo, Democratic Republic of the' >Congo, Democratic Republic of the</option><option value='Congo, Republic of the' >Congo, Republic of the</option><option value='Cook Islands' >Cook Islands</option><option value='Costa Rica' >Costa Rica</option><option value='Côte d&#039;Ivoire' >Côte d&#039;Ivoire</option><option value='Croatia' >Croatia</option><option value='Cuba' >Cuba</option><option value='Curaçao' >Curaçao</option><option value='Cyprus' >Cyprus</option><option value='Czech Republic' >Czech Republic</option><option value='Denmark' >Denmark</option><option value='Djibouti' >Djibouti</option><option value='Dominica' >Dominica</option><option value='Dominican Republic' >Dominican Republic</option><option value='Ecuador' >Ecuador</option><option value='Egypt' >Egypt</option><option value='El Salvador' >El Salvador</option><option value='Equatorial Guinea' >Equatorial Guinea</option><option value='Eritrea' >Eritrea</option><option value='Estonia' >Estonia</option><option value='Eswatini (Swaziland)' >Eswatini (Swaziland)</option><option value='Ethiopia' >Ethiopia</option><option value='Falkland Islands' >Falkland Islands</option><option value='Faroe Islands' >Faroe Islands</option><option value='Fiji' >Fiji</option><option value='Finland' >Finland</option><option value='France' >France</option><option value='French Guiana' >French Guiana</option><option value='French Polynesia' >French Polynesia</option><option value='French Southern Territories' >French Southern Territories</option><option value='Gabon' >Gabon</option><option value='Gambia' >Gambia</option><option value='Georgia' >Georgia</option><option value='Germany' >Germany</option><option value='Ghana' >Ghana</option><option value='Gibraltar' >Gibraltar</option><option value='Greece' >Greece</option><option value='Greenland' >Greenland</option><option value='Grenada' >Grenada</option><option value='Guadeloupe' >Guadeloupe</option><option value='Guam' >Guam</option><option value='Guatemala' >Guatemala</option><option value='Guernsey' >Guernsey</option><option value='Guinea' >Guinea</option><option value='Guinea-Bissau' >Guinea-Bissau</option><option value='Guyana' >Guyana</option><option value='Haiti' >Haiti</option><option value='Heard and McDonald Islands' >Heard and McDonald Islands</option><option value='Holy See' >Holy See</option><option value='Honduras' >Honduras</option><option value='Hong Kong' >Hong Kong</option><option value='Hungary' >Hungary</option><option value='Iceland' >Iceland</option><option value='India' >India</option><option value='Indonesia' >Indonesia</option><option value='Iran' >Iran</option><option value='Iraq' >Iraq</option><option value='Ireland' >Ireland</option><option value='Isle of Man' >Isle of Man</option><option value='Israel' >Israel</option><option value='Italy' >Italy</option><option value='Jamaica' >Jamaica</option><option value='Japan' >Japan</option><option value='Jersey' >Jersey</option><option value='Jordan' >Jordan</option><option value='Kazakhstan' >Kazakhstan</option><option value='Kenya' >Kenya</option><option value='Kiribati' >Kiribati</option><option value='Kuwait' >Kuwait</option><option value='Kyrgyzstan' >Kyrgyzstan</option><option value='Lao People&#039;s Democratic Republic' >Lao People&#039;s Democratic Republic</option><option value='Latvia' >Latvia</option><option value='Lebanon' >Lebanon</option><option value='Lesotho' >Lesotho</option><option value='Liberia' >Liberia</option><option value='Libya' >Libya</option><option value='Liechtenstein' >Liechtenstein</option><option value='Lithuania' >Lithuania</option><option value='Luxembourg' >Luxembourg</option><option value='Macau' >Macau</option><option value='Macedonia' >Macedonia</option><option value='Madagascar' >Madagascar</option><option value='Malawi' >Malawi</option><option value='Malaysia' >Malaysia</option><option value='Maldives' >Maldives</option><option value='Mali' >Mali</option><option value='Malta' >Malta</option><option value='Marshall Islands' >Marshall Islands</option><option value='Martinique' >Martinique</option><option value='Mauritania' >Mauritania</option><option value='Mauritius' >Mauritius</option><option value='Mayotte' >Mayotte</option><option value='Mexico' >Mexico</option><option value='Micronesia' >Micronesia</option><option value='Moldova' >Moldova</option><option value='Monaco' >Monaco</option><option value='Mongolia' >Mongolia</option><option value='Montenegro' >Montenegro</option><option value='Montserrat' >Montserrat</option><option value='Morocco' >Morocco</option><option value='Mozambique' >Mozambique</option><option value='Myanmar' >Myanmar</option><option value='Namibia' >Namibia</option><option value='Nauru' >Nauru</option><option value='Nepal' >Nepal</option><option value='Netherlands' >Netherlands</option><option value='New Caledonia' >New Caledonia</option><option value='New Zealand' >New Zealand</option><option value='Nicaragua' >Nicaragua</option><option value='Niger' >Niger</option><option value='Nigeria' >Nigeria</option><option value='Niue' >Niue</option><option value='Norfolk Island' >Norfolk Island</option><option value='North Korea' >North Korea</option><option value='Northern Mariana Islands' >Northern Mariana Islands</option><option value='Norway' >Norway</option><option value='Oman' >Oman</option><option value='Pakistan' >Pakistan</option><option value='Palau' >Palau</option><option value='Palestine, State of' >Palestine, State of</option><option value='Panama' >Panama</option><option value='Papua New Guinea' >Papua New Guinea</option><option value='Paraguay' >Paraguay</option><option value='Peru' >Peru</option><option value='Philippines' >Philippines</option><option value='Pitcairn' >Pitcairn</option><option value='Poland' >Poland</option><option value='Portugal' >Portugal</option><option value='Puerto Rico' >Puerto Rico</option><option value='Qatar' >Qatar</option><option value='Réunion' >Réunion</option><option value='Romania' >Romania</option><option value='Russia' >Russia</option><option value='Rwanda' >Rwanda</option><option value='Saint Barthélemy' >Saint Barthélemy</option><option value='Saint Helena' >Saint Helena</option><option value='Saint Kitts and Nevis' >Saint Kitts and Nevis</option><option value='Saint Lucia' >Saint Lucia</option><option value='Saint Martin' >Saint Martin</option><option value='Saint Pierre and Miquelon' >Saint Pierre and Miquelon</option><option value='Saint Vincent and the Grenadines' >Saint Vincent and the Grenadines</option><option value='Samoa' >Samoa</option><option value='San Marino' >San Marino</option><option value='Sao Tome and Principe' >Sao Tome and Principe</option><option value='Saudi Arabia' >Saudi Arabia</option><option value='Senegal' >Senegal</option><option value='Serbia' >Serbia</option><option value='Seychelles' >Seychelles</option><option value='Sierra Leone' >Sierra Leone</option><option value='Singapore' >Singapore</option><option value='Sint Maarten' >Sint Maarten</option><option value='Slovakia' >Slovakia</option><option value='Slovenia' >Slovenia</option><option value='Solomon Islands' >Solomon Islands</option><option value='Somalia' >Somalia</option><option value='South Africa' >South Africa</option><option value='South Georgia' >South Georgia</option><option value='South Korea' >South Korea</option><option value='South Sudan' >South Sudan</option><option value='Spain' >Spain</option><option value='Sri Lanka' >Sri Lanka</option><option value='Sudan' >Sudan</option><option value='Suriname' >Suriname</option><option value='Svalbard and Jan Mayen Islands' >Svalbard and Jan Mayen Islands</option><option value='Sweden' >Sweden</option><option value='Switzerland' >Switzerland</option><option value='Syria' >Syria</option><option value='Taiwan' >Taiwan</option><option value='Tajikistan' >Tajikistan</option><option value='Tanzania' >Tanzania</option><option value='Thailand' >Thailand</option><option value='Timor-Leste' >Timor-Leste</option><option value='Togo' >Togo</option><option value='Tokelau' >Tokelau</option><option value='Tonga' >Tonga</option><option value='Trinidad and Tobago' >Trinidad and Tobago</option><option value='Tunisia' >Tunisia</option><option value='Turkey' >Turkey</option><option value='Turkmenistan' >Turkmenistan</option><option value='Turks and Caicos Islands' >Turks and Caicos Islands</option><option value='Tuvalu' >Tuvalu</option><option value='Uganda' >Uganda</option><option value='Ukraine' >Ukraine</option><option value='United Arab Emirates' >United Arab Emirates</option><option value='United Kingdom' >United Kingdom</option><option value='United States' >United States</option><option value='Uruguay' >Uruguay</option><option value='US Minor Outlying Islands' >US Minor Outlying Islands</option><option value='Uzbekistan' >Uzbekistan</option><option value='Vanuatu' >Vanuatu</option><option value='Venezuela' >Venezuela</option><option value='Vietnam' >Vietnam</option><option value='Virgin Islands, British' >Virgin Islands, British</option><option value='Virgin Islands, U.S.' >Virgin Islands, U.S.</option><option value='Wallis and Futuna' >Wallis and Futuna</option><option value='Western Sahara' >Western Sahara</option><option value='Yemen' >Yemen</option><option value='Zambia' >Zambia</option><option value='Zimbabwe' >Zimbabwe</option></select> <label for='input_6_10_6' id='input_6_10_6_label' >Country</label> </span> <div class='gf_clear gf_clear_complex'></div> </div></li> </ul></div> <div class='gform_footer top_label'> <input type='submit' id='gform_submit_button_6' class='gform_button button' value='Sign Up' onclick='if(window["gf_submitting_6"]){return false;} window["gf_submitting_6"]=true; ' onkeypress='if( event.keyCode == 13 ){ if(window["gf_submitting_6"]){return false;} window["gf_submitting_6"]=true; jQuery("#gform_6").trigger("submit",[true]); }' /> <input type='hidden' class='gform_hidden' name='is_submit_6' value='1' /> <input type='hidden' class='gform_hidden' name='gform_submit' value='6' /> <input type='hidden' class='gform_hidden' name='gform_unique_id' value='' /> <input type='hidden' class='gform_hidden' name='state_6' value='WyJbXSIsIjNkNTNhNjhlOThlOGY4N2JlZWZiNTRmZDhlZDQ5OWI3Il0=' /> <input type='hidden' class='gform_hidden' name='gform_target_page_number_6' id='gform_target_page_number_6' value='0' /> <input type='hidden' class='gform_hidden' name='gform_source_page_number_6' id='gform_source_page_number_6' value='1' /> <input type='hidden' name='gform_field_values' value='' /> </div> </form> </div><script type='text/javascript'> jQuery(document).bind('gform_post_render', function(event, formId, currentPage){if(formId == 6) {gf_global["number_formats"][6] = {"1":{"price":false,"value":false},"2":{"price":false,"value":false},"7":{"price":false,"value":false},"20":{"price":false,"value":false},"15":{"price":false,"value":false},"19":{"price":false,"value":false},"22":{"price":false,"value":false},"10":{"price":false,"value":false}};if(window['jQuery']){if(!window['gf_form_conditional_logic'])window['gf_form_conditional_logic'] = new Array();window['gf_form_conditional_logic'][6] = { logic: { 10: {"field":{"actionType":"show","logicType":"any","rules":[{"fieldId":"22","operator":"is","value":"Yes, I love mail!"}]},"nextButton":null,"section":null} }, dependents: { 10: [10] }, animation: 1, defaults: {"1":{"1.2":"","1.3":"","1.4":"","1.6":"","1.8":""},"15":["choice_6_15_1"],"10":{"10.1":"","10.2":"","10.3":"","10.4":"","10.5":"","10.6":""}}, fields: {"1":[],"2":[],"7":[],"20":[],"15":[],"19":[],"22":[10],"10":[]} }; if(!window['gf_number_format'])window['gf_number_format'] = 'decimal_dot';jQuery(document).ready(function(){gf_apply_rules(6, [10], true);jQuery('#gform_wrapper_6').show();jQuery(document).trigger('gform_post_conditional_logic', [6, null, true]);} );} gformInitChosenFields('#input_6_7','No results matched');} } );jQuery(document).bind('gform_post_conditional_logic', function(event, formId, fields, isInit){gformInitChosenFields('#input_6_7','No results matched');} );</script><script type='text/javascript'> jQuery(document).ready(function(){jQuery(document).trigger('gform_post_render', [6, 1]) } ); </script>
MENU