Nailing the diagnosis pathology. Veterinary consultancy content from SASH

1. Nailing the diagnosis
Help your pathology help you
Small Animal Specialist Hospital
Dr Sophia Tzannes

2. Nailing the diagnosis
Help your pathology help you
• Cytology
• When, how, what and where?
• Advanced cytology and additional tests
• Histopathology
• Pre-op, post-op- when does it matter?
• When do we ask for immunohistochemistry?

3. Nailing the diagnosis
Help your pathology help you
• ‘Hugo’
• ‘Gywna’
• ‘Forster’
• ‘Dudley’

4. CYTOLOGY
• When?
• How?
• What?
• Where?

5. CYTOLOGY:
WHEN
Pre-surgical diagnosis is encouraged
• Owners expectations: prognosis and financial considerations
• Determine whether further information is required prior definitive
surgery
• Staging
• May result in non-surgical treatment as best option
• Not considered a substitute for histopathology

6. CYTOLOGY:
WHEN
Staging and monitoring when diagnosis known
• Staging
• Monitor for recurrence of lesions cytologically

7. CYTOLOGY
HOW
• Make sure what you are sampling is what you wish to sample
• Make sure what you have sampled is of diagnostic quality
• repeat multiple times if necessary, use different techniques
• Make sure that you submit an adequate number of slides

8. CYTOLOGY
HOW
fat
lesion
• Make sure what you are
sampling is what you
wish to sample
• ‘Hugo’ is a 12 y MN Rottweiler X
• Presented August 2014 for oncological
and surgical assessment of an infiltrating
lipoma
• Fine needle aspirates by referring vet had
consistently revealed droplets of free fat
and adipocytes
• CT performed to evaluate pre-operatively

9. CYTOLOGY
HOW:
Make sure what you are sampling is what you wish to sample
• Visualisation is the key
• Superficial lesions: estimate trajectory
• Deep lesions: consider imaging as guidance

10. CYTOLOGY:
HOW
Make sure what you have sampled is of diagnostic quality
9y FN Irish Setter
Presented with mammary masses
Vulval mass detected during prep
Fine needle aspirate (FNA)

11. CYTOLOGY:
HOW : MAKE SURE WHAT YOU HAVE SAMPLED IS OF DIAGNOSTIC QUALITY
• Equipment needed
• Common problems encountered with slide preparation:
• Blood contamination
• Low cellularity
• Cell rupture or lysis
• Smear too thick
• Artefacts

12. CYTOLOGY:
HOW : MAKE SURE WHAT YOU HAVE SAMPLED IS OF DIAGNOSTIC QUALITY
• Common problems encountered with slide preparation:
• Blood contamination
• Low cellularity
• Cell rupture or lysis
• Smear too thick
• Artefacts
-Lymph nodes
-Mast cell tumours
-Internal organs
Liver, spleen
-Vascular tumours
-Ulcerated lesions
-Mucous membranes
Spindle cell tumours
Easy to exfoliate:
Round cell tumours
+/- epithelial cells

13. CYTOLOGY:
HOW : MAKE SURE WHAT YOU HAVE SAMPLED IS OF DIAGNOSTIC QUALITY
• Common problems encountered with slide preparation:
• Blood contamination
• Low cellularity
• Cell rupture or lysis
• Smear too thick
• Artefact

14. CYTOLOGY:
HOW : MAKE SURE WHAT YOU HAVE SAMPLED IS OF DIAGNOSTIC QUALITY
1. Is the slide of diagnostic quality?
2. Are there different types of cells present?
3. Are inflammatory cells present?
4. Can you identify the cells belonging to the tissue of origin?
5. Unusual cells?
a. Shape
b. Cluster or occur individually
c. Cytoplasmic detail and nuclear detail
6. Extra cellular material/infectious agents

15. CYTOLOGY:
WHAT IS IT?
Is the slide of
diagnostic quality?
Yes
Are there different
types of cells
present?
Yes
Are inflammatory cells
present?
Yes
Can you identify the
cells belonging to
the tissue of origin?
Yes, epithelial cells
Unusual cells? No
Extra cellular
material/infectious
agents
No

16. CYTOLOGY:
WHAT IS IT?
Is the slide of
diagnostic quality?
Yes
Are there different
types of cells
present?
Yes
Are inflammatory
cells present?
Yes
Can you identify the
cells belonging to
the tissue of
origin?
Yes, epithelial
cells
Unusual cells? No
Extra cellular
material/infectious
agents
No
Inflammatory
Neoplastic
Degenerative

17. CYTOLOGY:
WHAT IS IT?
Inflammatory
Neoplastic
Degenerative
MCT: Toluidine blue

18. CYTOLOGY:
WHAT IS IT?
Neoplastic
• Cytological criteria of malignancy:
• Nuclear criteria
• e.g.: variation in nuclear size and shape- multinucleation, especially if nuclei within same cell vary in size;
increased nuclear:cytoplasmic ratio or variation within same population of cells; coarse and ropey chromatin
pattern; nuclear moulding; increased number of mitotic figures; variation in nucleolar size, shape and
number, especially within same nucleus- bizarre forms,
• Cellular criteria
• e.g. variation in cell size and shape (greater in malignancy), cell population in abnormal location
• Cytoplasmic criteria
• e.g. cytoplasmic basophilia, vacuolation and granulation

19. CYTOLOGY:
WHAT IS IT?
Neoplastic

20. CYTOLOGY:
WHAT IS IT?
Is the slide of
diagnostic quality?
Yes
Are there different
types of cells
present?
Yes
Are inflammatory
cells present?
Yes
Can you identify the
cells belonging to
the tissue of
origin?
Yes, epithelial
cells
Unusual cells? No
Extra cellular
material/infectious
agents
No
Inflammatory

21. CYTOLOGY:
WHAT IS IT?
• Limitations
• Mammary > histopathology
• Cannot grade
• May not be definitive

22. CYTOLOGY: WHERE?
WHERE CAN WE SAMPLE, WHERE SHOULD WE SAMPLE?
• External tissues
• Internal tissues and fluid
• Contra-indications include:
Vascular lesion
Coagulopathy
Impaired access
Hollow organ
Cancer seeding (TCC)

23. CYTOLOGY: WHERE?
WHERE CAN WE SAMPLE, WHERE SHOULD WE SAMPLE?
• Staging
• Performed on patients with malignant lesions
• Knowing pattern of metastatic spread is helpful
• Less differentiated MCT recommend lymph node, liver and spleen aspirates
• Soft tissue sarcoma: haematogenous route to lungs (other soft tissue, liver) common
• Subtype dependent: histiocytic/synovial cell sarcoma to regional LN (plus lung, etc)
• Carcinoma: lymphatic route

24. CYTOLOGY: WHERE CAN WE SAMPLE, WHERE SHOULD WE SAMPLE?
CANINE LYMPHOMA- SPECIAL CONSIDERATIONS
• Multi-centric lymphoma
• Which lymph nodes?
• How many samples?
• What should I be requesting to maximise my diagnosis?

25. • Immunocytochemistry and immunohistochemistry refer to
the process of detecting antigens in tissues or cells by
exploiting the antigen-antibody binding process found in
biological tissues.
CYTOLOGY:
LYMPHOMA- SPECIAL CONSIDERATIONS
IMMUNOPHENOTYPING AND FLOW CYTOMETRY

26. IMMUNOPHENOTYPING
• The diversity of immunophenotyping markers used in
diagnostic pathology is substantial and more markers are
becoming available in the veterinary field. Some examples of
commonly used markers include:
– CD3: T cell lymphoma
– CD79a: B cell lymphoma
– CD18, 11: Histiocytic disease
– CD117: KIT used to identify MCT’s and GIST’s
– Vimentin: a marker common to sarcomas

27. PARR AND FLOW
What is the difference between flow cytometry and PARR?
• The PARR assay is a PCR assay in which we are amplifying DNA to evaluate lymphocyte
(LCT) receptor gene length. In a heterogenous LCT population the LCT’s are all genetically
distinct so the LCT receptor genes vary in length.
• Homogenous or clonal LCT population, is often malignant and have identical gene length
throughout the population.
• The results tell us if the majority of cells in the sample
are derived from the same original clone (most
consistent with neoplasia), or from multiple clones
(most consistent with a reactive process- inflammation,
immune mediated disease or infection)

28. PARR AND FLOW
What is the difference between flow cytometry and PARR?
• Flow cytometry (FC) allows identification and quantitation of cell surface
markers
• The FC study involves staining live cells (blood, BM, LCT) with labelled
antibodies that bind to proteins expressed on the cell surface
• The cells are analyzed on a flow cytometer, which tells us how many cells of each type are
present. This information allows us to determine the lineage of the cells present, and
whether they are homogeneous (more consistent with neoplasia) or heterogeneous (more
consistent with a reactive process
• Limitation – sample collection- appropriate cytofixative for FNA samples (48 hours), or
blood/bone marrow into EDTA (24 hours)

29. CYTOLOGY:
CANINE LYMPHOMA- SPECIAL CONSIDERATIONS
FLOW CYTOMETRY
• Immunophenotyping lymphoma
• Prognosis
• Treatment guidance
CD3 (T cell marker)
CD4 (helper T cells)
CD5 (T cell marker)
CD8 (cytotoxic T cells)
CD21 (B cells, but not their precursors)
CD34 (stem cell marker, expressed in acute
leukaemia)
CD3-e (pan T cell marker)
CD79a (pan B cell marker)
Myeloperoxidase (expressed by myeloblasts
and granulocyte precursors)
MAC387 (expressed by monoblasts, and
some granulocyte precursors)
CD14 (expressed by monoblasts and
monocytes)

30. CYTOLOGY:
CANINE LYMPHOMA- SPECIAL CONSIDERATIONS
FLOW CYTOMETRY
• Differentiation of hyperplasia and neoplastic populations
• Characterisation of leukaemias CD3 (T cell marker)
CD4 (helper T cells)
CD5 (T cell marker)
CD8 (cytotoxic T cells)
CD21 (B cells, but not their precursors)
CD34 (stem cell marker, expressed in acute
leukaemia)
CD3-e (pan T cell marker)
CD79a (pan B cell marker)
Myeloperoxidase (expressed by myeloblasts
and granulocyte precursors)
MAC387 (expressed by monoblasts, and
some granulocyte precursors)
CD14 (expressed by monoblasts and
monocytes)

31. CYTOLOGY:
CANINE LYMPHOMA- SPECIAL CONSIDERATIONS
PARR
• Sample factors:
• cell lysis/poor preservation
• low diagnostic yield
• location of sample- e.g. intestinal tract
• Disease factors:
• equivocal diagnosis- ‘reactive node’
• early diagnosis
• certain lymphoma sub-types versus
• persistent lymphocytosis

32. HISTOPATHOLOGY
• Pre-op, post-op- when does it matter?
• When do we ask for immunohistochemistry?

33. HISTOPATHOLOGY
• Definitive diagnosis
Punch
Tru-cut
Grab/pinch
Incisional biopsy

34. HISTOPATHOLOGY
Pre-op, post-op- when does it matter?
• Pre-operative histopathology especially important when
grading may influence surgical plan
• Surgical margins
Grade 1 and 2:
Minimum 2 cm and one deep
fascial plane
Grade 3:
Minimum 3 cm and one deep
fascial plane
Mast cell tumour, H+E

35. HISTOPATHOLOGY
Pre-op, post-op- when does it matter?
• Pre-operative histopathology especially important when
grading may influence surgical plan
• Surgical approach when surgery may be challenging:
• large
• localisation

36. HISTOPATHOLOGY
Pre-op, post-op- when does it matter?
• Pre-operative histopathology is not indicated where there
is no influence on surgical plan
• Location
• renal
• spleen
• mammary
Always obtain
post-operative
histopathology

37. HISTOPATHOLOGY:
When do we ask for immunohistochemistry?
• Diagnosis is inconclusive
• Diagnosis does not fit clinical picture
• Where a more precise diagnosis will influence
prognosis or treatment
•Round cell neoplasia
•Fibrohistiocytic splenic neoplasia
•Intestinal neoplasia (sarcoma, other)

38. HISTOPATHOLOGY:
When do we ask for immunohistochemistry?
•‘Forster’, 12y MN Border Collie
•Presented as a referral for a recurrent oral
melanoma
Forster’s biopsy:
Round cell
population

39. • Further diagnosis with
histopathology and
immunohistochemistry
Epitheliotropic T cell lymphoma
Amelanotic melanoma
Round cells
Histiocytic cells
Mast cells
Lymphoma
Plasma cells
TVT
(Melanoma)
HISTOPATHOLOGY:
When do we ask for immunohistochemistry?

40. Lip mass. Extensively infiltrating through the submucosa are myriad
round cells with scant amounts of lightly eosinophilic to clear
cytoplasm and no obvious pigment. The cells have atypical lymphoid
appearance with round nuclei containing granular to branched chromatin
and 1 nucleolus. Some of the nuclei are indented while occasional cells
have a slightly larger nucleus. The cells show aggregation around
vessels as well as infiltration into bundles of nerve and skeletal
muscle. The mitotic index is 5. There are scattered interstitial
eosinophils, small dark lymphocytes, occasional plasma cells and
macrophages. Within the overlying haired skin is a similar population of
round cells showing epitheliotropism, and also affecting hair follicles,
sebaceous glands and sweat glands. These cells often have clear
cytoplasm and infiltrate epithelium as individual cells and small
clusters. Affected regions of the epithelium are subtended by plasma
cells, small dark lymphocytes and a few of the atypical lymphocytes.
Distribution of this change within the affected haired skin is not
uniform. The mucosal surface is affected but to a lesser extent.
Neoplastic cells are not seen at mucosal and haired skin margins.
IMMUNOHISTOCHEMISTRY REPORT
The neoplastic lymphocytes within epithelium and deeper tissue are
strongly and uniformly labelled with CD3
Lip mass. Extensively infiltrating through the submucosa are myriad
round cells with scant amounts of lightly eosinophilic to clear
cytoplasm and no obvious pigment. The cells have atypical lymphoid
appearance with round nuclei containing granular to branched chromatin
and 1 nucleolus. Some of the nuclei are indented while occasional cells
have a slightly larger nucleus. The cells show aggregation around
vessels as well as infiltration into bundles of nerve and skeletal
muscle. The mitotic index is 5. There are scattered interstitial
eosinophils, small dark lymphocytes, occasional plasma cells and
macrophages. Within the overlying haired skin is a similar population of
round cells showing epitheliotropism, and also affecting hair follicles,
sebaceous glands and sweat glands. These cells often have clear
cytoplasm and infiltrate epithelium as individual cells and small
clusters. Affected regions of the epithelium are subtended by plasma
cells, small dark lymphocytes and a few of the atypical lymphocytes.
Distribution of this change within the affected haired skin is not
uniform. The mucosal surface is affected but to a lesser extent.
Neoplastic cells are not seen at mucosal and haired skin margins.
IMMUNOHISTOCHEMISTRY REPORT
The neoplastic lymphocytes within epithelium and deeper tissue are
strongly and uniformly labelled with CD3
• IMHC: CD3 +(CD8+)
HISTOPATHOLOGY:
When do we ask for immunohistochemistry?
Epitheliotropic
lymphoma

41. • Histopathology
• 50% (7/14) of oral cases were conclusively diagnosed
with histopathology alone (Nemec et al, 2012)
• early stages can have marked mixed inflammatory
infiltrate, late stages > B lymphocytes
• >50% there is a mix of lymphocyte cell size reported
• IMHC
• humans: 5-10% lymphoid neoplasia cannot be
diagnosed with use of histopathology and IMHC
• PARR
• T cell receptor (TCR) gamma rearrangement
• Sensitivity 80-95%, Specificity 96-100%
Clinical features
Histopathology
Immunohistochemistry
Clonality testing
Clinical follow up and
repeat biopsy
HISTOPATHOLOGY:
When do we ask for immunohistochemistry?
Epitheliotropic lymphoma: diagnosis

42. HISTOPATHOLOGY:
When do we ask for immunohistochemistry? Diagnosis
Change in diagnosis
Change in management
Surgical > Medical

43. HISTOPATHOLOGY:
When do we ask for immunohistochemistry?
•‘Dudley’, 12y MN Staffordshire Bull Terrier
•Presented as a referral for haemabdomen
•Diagnosed with splenic mass
•Splenectomy after staging
•Diagnosis: splenic sarcoma
The splenic masses reflect
a malignancy which
consists of variably
pleomorphic spindloid to
histiocytic cells admixed
with lymphoid nodules and
extramedullary
haematopoiesis, consisting
with a fibrohistiocytic
nodule.
The regions of necrosis and
areas with a relatively high
mitotic rate warrants a
diagnosis of malignancy.
Diagnosis: splenic sarcoma

44. HISTOPATHOLOGY:
When do we ask for immunohistochemistry?
•Immunohistochemistry requested
•Diagnosis: fibrohistiocytic sarcoma
Vimentin
Desmin
Smooth muscle actin
Factor VIII
CD3
CD79a
CD18

45. • Histologic and immunohistochemical review of splenic fibrohistiocytic nodules (SFHN) in dogs (Moore
2012)
• Splenic FHN cases were re-evaluated in 32 dogs
• Histopathology Grade I(2) Grade II (9) Grade III(21) dogs
• CD3, CD20, CD79a, CD18, CD11d, K1-67 used to reclassify
• Grade I- MZL and lymphoid nodular hyperplasia
• Grade II-MZ hyperplasia(1), MZL (1), complex hyperplasia (2), LNH(1), stromal sarcoma (3)
• Grade III- MZH(1), diffuse large B cell LSA(1), MZL(1)CNH (6),LNH (1), SS (5), HS (6)
• Prognosis:
HISTOPATHOLOGY:
When do we ask for immunohistochemistry? Prognosis
HS- poor 74 days
Stromal sarcoma 488 days, 56% alive 1 year
CNH MST 387 days 70% alive 1 year
LNH MST 570 days 60% alive 2 years

46. HISTOPATHOLOGY:
When do we ask for immunohistochemistry? Therapy
• Intestinal tumours
• Reclassification of small intestinal and cecal smooth
muscle tumours in 72 dogs (Maas et al, 2007)
• Retrospective study of 47 dogs with a prior diagnosis of
leiomyoma or leiomyosarcoma
• 85% reclassified as gastrointestinal stromal tumour
(GIST) or GIST-like
• Therapeutic relevance
• GIST + c-kit, target for tyrosine kinase inhibitors (TKIs)
• Diagnosis is inconclusive
• Diagnosis does not fit clinical picture
• Where a more precise diagnosis will influence prognosis ortreatment
•Round cell neoplasia
•Fibrohistiocytic splenic neoplasia
•Intestinal neoplasia (sarcoma, other)

47. HISTOPATHOLOGY:
When do we ask for immunohistochemistry? Therapy
• Mast cell tumours
• Ki-67 prognostic, may elect adjunctive treatments if high
• KIT (CD 117) if use of TKIs considered
C-Kit staininghigh Ki-67low Ki-67

48. HISTOPATHOLOGY:
CANINE LYMPHOMA- SPECIAL CONSIDERATIONS
• Summary of Canine Malignant Lymphoma Revised From the Revised European-American Classification of
Lymphoid Neoplasms/ World Health Organization Classification of Lymphoid Neoplasms
B Cell Neoplasms
Precursor B cell neoplasms
Precursor B lymphoblastic leukemia/lymphoma
Mature (peripheral) B cell neoplasms
B cell chronic lymphocytic leukemia/prolymphocytic
Leukemia/small lymphocytic lymphoma
B cell prolymphocytic leukemia
Lymphoplasmacytic lymphoma
Splenic marginal zone B cell lymphoma
Plasma cell myeloma/plasmacytoma
Extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue type
Nodal marginal zone lymphoma
Follicular lymphoma
Mantle cell lymphoma
Diffuse large B cell lymphomaa
Mediastinal large B cell lymphoma
Burkitt’s lymphoma/Burkitt’s cell leukemia
Provisional entity: high-grade B cell lymphoma
Burkitt’s-likea
Primary effusion lymphoma
T Cell and Putative Natural Killer Cell Neoplasms
Precursor T cell neoplasm
Precursor T lymphoblastic
Lymphoma/leukemia
Mature (peripheral) T cell and natural killer cell neoplasms
T cell prolymphocytic leukemia
Large granular lymphocyte leukemia (LGL)
Aggressive natural killer (NK) cell leukemia
Peripheral T cell lymphomas, unspecifieda
Adult T cell lymphoma/leukemia
Intestinal T cell lymphoma (+enteropathy associated)
Hepatosplenic gdT cell lymphoma
Subcutaneous panniculitis-like T cell lymphoma
Mycosis fungoides/Sezary syndrome
Anaplastic large cell lymphoma, T and null cell primary cutaneous
type
Peripheral T cell lymphoma not otherwise specified
Angioimmunoblastic T cell lymphoma
Angiocentric T cell lymphoma
a Peripheral T cell lymphomas are those that are not otherwise specified
(NOS) to a specific subtype by further definition
Personalised
lymphoma
treatment

49. • Lymphomas divided into 3 major groups
• high, intermediate, low grade
• Most common lymphoma was centroblastic large B cell
CANINE LYMPHOMA- SPECIAL CONSIDERATIONS
(Valli et al, 2010)

50. HISTOPATHOLOGY:
CANINE LYMPHOMA- SPECIAL CONSIDERATIONS
(Valli et al, 2010)
Personalised
lymphoma
treatment
• Does the specific diagnosis of lymphoma subtype have a similar impact on survival time in
dogs as it did in humans?
Low grade T cell (T zone)
lymphoma
Longest survival time
(622 days)
T cell high grade
(peripheral T cell)
lymphoma
Shortest survival time
(162 days)
Clinical signs
Low grade often none
High grade frequently
unwell

51. HISTOPATHOLOGY:
CANINE LYMPHOMA- SPECIAL CONSIDERATIONS
(Valli et al, 2010)
Personalised
lymphoma
treatment
• Does a specific diagnosis in a dog influence the chemotherapy required?
High Grade B and T cell
lymphoma
Effective chemotherapy
increased survival time
T zone lymphoma
Chemotherapy decreased
survival time
(13 dogs with no
treatment had longest
survival of 687 days)

52. • Indolent lymphoma comprises 5- 29% of all canine lymphomas
• Limited information regarding the subtypes and biological behavior.
• Canine indolent lymphoma consists of a group of diseases that are histopathologically
similar to subtypes of non-Hodgkin’s lymphoma identified in people.
• Histopathological subtypes:
B cell- marginal zone (MZL), follicular lymphoma
(FL) and mantle cell lymphoma (MCL ),
T-zone lymphoma (TZL).
HISTOPATHOLOGY:
CANINE LYMPHOMA- SPECIAL CONSIDERATIONS
(Valli et al, 2010)

53. • B cell predominant
• Both B and T cell indolent lymphomas share a low mitotic rate,
slow rate of progression
• TZL can be difficult to recognize as malignancy given the small
mature –appearing cell type and low mitotic activity.
• Detection of clonality is useful adjunct to histologic
examination
HISTOPATHOLOGY:
CANINE LYMPHOMA- SPECIAL CONSIDERATIONS
INDOLENT LYMPHOMA

54. Clinical features
Histopathology
Immunohistochemistry
Clonality testing
Clinical follow up and
repeat biopsy
NAILING THE DIAGNOSIS: MAKING THE MOST OF OUR PATHOLOGY
Cytology
• Make sure what you are sampling is what you wish to sample
• Make sure what you have sampled is of diagnostic quality
• Make sure that you submit an adequate number of slides
• Diagnosis is inconclusive
• Diagnosis does not fit clinical picture
• Where a more precise diagnosis will influence prognosis ortreatment
•Round cell neoplasia
•Immunophenotyping
•Clonality testing
•Fibrohistiocytic splenic neoplasia
•Intestinal neoplasia (sarcoma, other)