The following tests are not included in routine panels and must be ordered separately.

• ​Bile acids: Fasted or paired with post-prandial (order fasted and post-prandial BA)
• Ionized Ca Panel (i-Ca): iCa, Na, K, Cl. We have found that samples mailed in for blood gas/ionized calcium analysis are unreliable and do not recommend mailing in samples for this testing.
• Ethylene glycol
• Fructosamine​

Cytology

Cytology focuses on the investigation of disease processes by examining tissue samples microscopically. Cells are evaluated individually under high magnification with the purpose of diagnosing types of inflammation, infectious agents, types of cancer, or other tissue abnormalities. Often, cytology can guide the next diagnostic step in patient evaluation.

Digital Photos With Cytology Specimens 

When submitting specimens for cytological examination, we encourage you to include digital photos of any pertinent anatomical features that may aid our pathologists in making their diagnosis. Email the following information so we can match your pictures with the specimen to clinpath@colostate.edu​. 

• ​Date
  
• Owner Name 
• Animal Name 
• Veterinarian and Clinic Name 
• Type of Sample 
• Phone and Fax Numbers
• Brief pertinent history and lesion description(s).

Cytochemical Special Stains

Cytochemical staining is a general term for a group of stains that detect specific chemical features. These can be performed on unstained or previously Romanowski stained slides and add information that cannot be easily confirmed by routine cytology staining. The following tests are performed in the laboratory:

Results: All cytochemical staining submissions must have routine cytology to confirm that the slides are appropriate for evaluation. Results will include the routine cytology description, description of the cytochemical staining, and interpretation of the case. 

Available tests interpreted by the clinical pathology laboratory 

These tests can be added to cytology evaluation. The staining is performed in the CSU VDL histopathology laboratory and evaluated by clinical pathologists.

• ​Acid Fast (Ziehl-Neelson): Carbol-Fuschia strongly binds to mycolic acid and the lipid cell wall of some organisms to produce distinct fuschia staining. This stain is most often used for Mycobacterium species and other bacteria. Charged as a special stain add-on.
• Alizarin Red S: Calcium chelates Alizarin Red S to produce a bright red stain.  This stain is used to confirm the presence of calcium (bone, Gamna-Gandy Bodies, etc). Charged as a special stain add-on.
• Congo Red Congo red stain binds the β-pleated sheets found in amyloid and produces a birefringence when viewed with polarized light.  This stain can be used to identify amyloid.  We do not currently perform potassium permanganate digestion for the distinction of amyloid types. Charged as a special stain add-on.
• GMS (Grocott Methenamine Silver) Aldehyde groups reactive with silver nitrate to produce metallic silver, which appears a brown-black.  This stain is most often used to highlight the presence of fungal elements and can detect other components, including neutrophils and some debris. Charged as a special stain add-on.
• PAS (Periodic acid-Schiff) Periodic acid is used to produce aldehydes in glycoproteins; the aldehydes react with fuchsin to produce a pink-magenta color.  This stain is used commonly to identify mucin, glycogen, fungal elements, and other structures. Diastase digestion prior to PAS staining is available. Charged as a special stain add-on.
• Von Kossa The von Kossa reaction binds silver to phosphorous, producing a brown-black labeling.  Because this typically occurs as a replacement of calcium, the stain is typically considered a stain for calcium. Charged as a special stain add-on.​

Protein Electrophoresis and Immunofixation 

Protein electrophoresis and immunofixation allow a more detailed evaluation of the protein profile than a biochemical total protein, albumin, and globin. These tests help to confirm the presence of an immunoglobulin-secreting tumor and other protein changes. Electrophoresis must be performed on the same sample at CSU if immunofixation is performed. 

• ​Protein electrophoresis uses classic techniques involving agarose gel and an electrical current to stratify proteins based on their size and charge. Because the technique is not species-specific proteins, it can be performed on samples from many species, including domestic and exotic species. Protein electrophoresis is typically performed on serum or urine. 
• ​Serum protein electrophoresis (SPE): Serum proteins are typically divided into albumin, alpha 1, alpha 2, beta 1, beta 2, and gamma globulin fractions. Specific proteins are found in each fraction and the electrophoretic pattern can be helpful in diagnosis a specific disease or disease process, see below. 
• Urine protein electrophoresis (UPE): Significant glomerular and tubular disease can allow serum proteins to pass into the urine, these can be detected with urine protein electrophoresis.   Free immunoglobulin light chains are small enough to pass through the renal glomeruli and into the urine; free light chains may be detectable by electrophoresis of concentrated urine.
• M-protein quantification: Monoclonal immunoglobulin protein (M-protein) will be quantified and reported separately when it can be confidently identified by routine serum or urine protein electrophoresis. Immunofixation may be needed to confirm that a suspected restricted protein band is an M-protein. 
• ​Immunofixation allows specific detection of immunoglobulin heavy and light chains and increases the sensitivity of SPE alone for the detection of monoclonal gammopathy by about 16%. It operates on similar principles to routine electrophoresis, but the gel is labeled to identify specific proteins.  Immunofixation can be used to classify the type of immunoglobulin in a monoclonal gammopathy or confirm that a high concentration, restricted protein band is an immunoglobulin. Protein electrophoresis must be performed on the sample at CSU prior to immunofixation; the combination of protein electrophoresis and immunofixation is a powerful tool that can be very helpful in challenging cases. Immunofixation can be performed on dog and cat samples using our routine antibody set or a set that identifies free light chains. 
• Routine immunofixation: Canine routine immunofixation evaluates for IgG, IgG4, IgA, and IgM immunoglobulin heavy chain and bound light chains.3 Feline routine immunofixation evaluates for IgG, IgA, and IgM immunoglobulin heavy chain and bound light chains.  We do not routinely perform this test on other species.
• Free light chain immunofixation: Detects immunoglobulin light chains that are not part of a complete immunoglobulin in the serum (serum-free light chains) or urine (Bence-Jones proteinuria). Current data suggests very good specificity for the identification of free light chains,  but sensitivity is unknown. Can be performed on dog and cat serum and urine.

When looking for complete monoclonal/biclonal gammopathy in the serum: 

• ​If ↑ serum TP --> SPE. Add routine IF if needed for detection or characterization of the M-protein
• If N serum TP --> SPE and routine IF 

• ​UPE combined with SPE or fLC IF of the urine
• SPE and fLC IF of the serum. This currently has lower sensitivity for detection.

• ​SPE/UPE until M-protein not apparent then IF to evaluate for complete response 

• UPE and SPE

Expected SPE results

Monoclonal/biclonal gammopathy. Monoclonal plasma cells and lymphocytes typically produce a single immunoglobulin clone (called an M-protein) resulting in a monoclonal gammopathy.  Monoclonal gammopathies are seen as a narrow, distinct band in the electrophoresis with labels with a single immunoglobulin class by immunofixation. Many monoclonal gammopathies dimerize and produce a biclonal appearance and we have detected truly biclonal gammopathies (i.e., production of 2 different heavy chain classes in the same patient).

A monoclonal gammopathy can produce significantly high total protein and globulin concentrations that alter plasma viscosity or interfere with clotting. Monoclonal gammopathy can also occur with low concentrations of serum M-protein that do not increase the serum total protein and globulin concentration. Current data suggests that about 30% of patients with a monoclonal gammopathy will have a total protein concentration within reference intervals and that about 10% of patients with a monoclonal gammopathy will have a globulin concentration within normal limits; immunofixation is especially helpful in detecting these low concentration M-proteins.6–8  Addition of routine and free light chain immunofixation to routine serum protein electrophoresis increases the sensitivity for detection of M-proteins from about 66% to >90%. This is especially true for serum-free light chains, which typically have a relatively normal SPE profile.

Documentation of a monoclonal gammopathy or Bence-Jones proteinuria is used to help make the diagnosis of multiple myeloma.  Monoclonal gammopathy can also be present in other myeloma-related disorders such as cutaneous or non-cutaneous extramedullary plasma cell tumors and some types of lymphoma. Increased concentrations of serum-free light chains are nephrotoxic and increase the risk of renal insufficiency. 

Change in M-protein concentration is used to monitor response to treatment in human medicine.  The human guidelines for response are: 

​Category	​​Serum-based Criteria
​CR - Complete Response​	​M-protein: Lack of M-protein by electrophoresis and immunofixation RID or Globulin: Within reference interval​​
​VGPR - Very Good Partial Response	​M-protein: > 90% reduction by electrophoresis but M-protein present by immunofixation RID or Globulin: >​​ 90% reduction and above upper reference limit
​PR - Partial Response	> 50% reduction
​MR - Minimal Response	> 25% reduction. Absolute decrease ​> 0.5 g/dl
SD - ​Stable Disease	​< 25% reduction or increase
​PD - Progressive Disease	​>​​ 25% increase from lowest value and    - If lowest value < 5/dl, Absolute increase > 0.5 g/dl​    - If lowest value >​​ 5g/dl, Absolute increase >​​ 1.0 g/dl

 

These criteria have been evaluated in the dog with the better response being associated with longer survival. Typically, monitoring M-protein concentration will involve SPE or UPE with immunofixation on the initial sample to positively identify and quantify the M-protein. As long as the M-protein can be detected by protein electrophoresis alone, subsequent M-protein quantification does not require immunofixation. When the M-protein cannot be detected by protein electrophoresis, immunofixation will be recommended to confirm the complete response and resolution of the monoclonal gammopathy.

Restricted polyclonal gammopathy/Oligoclonal gammopathy: If the immune reaction includes a strong response against a limited number of antigens, electrophoresis will have one or multiple restricted/monoclonal-appearing bands within a polyclonal background.  We have documented these restricted polyclonal gammopathies in cases with Ehrlichiosis, Leishmaniasis, Babesiosis, and other infectious and inflammatory diseases, including IgG4-related disease (IgG4-RD).10  IgG4-RD is a well-described human auto-inflammatory disease that has been documented in dogs.  The disease has multiple manifestations with a common thread of increased serum IgG4 concentration, plasmacytosis, fibroplasia, and eosinophilic inflammation. The restricted electrophoretic pattern of IgG4-RD and plasmacytosis can lead to a misdiagnosis of a monoclonal gammopathy and multiple myeloma; immunofixation can identify the restricted pattern as IgG4 and aid in the diagnosis of IgG4-RD.  Distinguishing between a monoclonal gammopathy and a restricted polyclonal gammopathy can sometimes be very challenging and require the combination of SPE, IF, the clinical history, and additional diagnostics. 

Polyclonal gammopathy: When inflammation, infection, or immune stimulation persist for an extended time course, multiple types of immunoglobulins are produced. This is seen as a broad increase in the beta 2 and gamma globulin region. Most polyclonal gammopathies are associated with infectious and inflammatory causes. 

Acute-phase protein response: Inflammation stimulates the production of positive acute-phase proteins, such as haptoglobin, ceruloplasmin, serum amyloid A and C-reactive protein. These proteins are typically found in the alpha and beta globulin fractions. Inflammation also is associated with decreased amounts of negative acute-phase proteins, most notably albumin. While this pattern helps explain the pathologic process, it is not specific for the cause of inflammation. 

Normal: Typical serum contains a predominance of albumin with lower amounts of various globulin subsets.​

Expected UPE results

Tubular Proteinuria is diagnosed when the UPE pattern suggests loss of smaller globulins and retention of albumin and larger globulins by the glomerulus.  Evaluation of the urine protein: creatine ratio can help distinguish pathologic or physiologic causes of tubular proteinuria. 

Glomerular Proteinuria is diagnosed when the UPE pattern suggests loss of albumin, transferrin, and other larger globulins through the glomerulus with retention of smaller globulins by the tubules. Evaluation of the urine protein: creatine ratio can help distinguish pathologic or physiologic causes of glomerular proteinuria. Correlation with the UPS is recommended.

Mixed Proteinuria is diagnosed when the UPE pattern suggests both tubular and glomerular protein loss.  Often these samples look identical to the serum.  Evaluation of the urine protein: creatine ratio can help distinguish pathologic or physiologic causes of mixed proteinuria. 

Bence-Jones Protienuria is used to indicate the presence of free immunoglobulin light chains in the urine.  This is highly suggested when there is a restricted protein band in the UPE that is not found in the SPE and can be confirmed by fLC IF.