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Clinical Pathology


Sample Collection and Handling

 

General points

  • Label all samples with the patient identification, date, and site sampled. Use pencil to label slides because ink comes off on our stainer. Use ink to label tubes. Please do not place adhesive labels on the slides as they affect our ability to stain them properly with our automated stainer.
    • Smears: If smears from more than one site are submitted, e.g. submandibular and popliteal lymph nodes, label each slide as to which site they represent.  This is important!! If the slides are not identified by site and the cytologic results are different, we will not be able to determine which site corresponds to which cytology.
    • Fluids: For any fluid other than peripheral blood, indicate what fluid is in the tube, e.g. bile, peritoneal (PTF), etc.
  • Submit all prepared smears.
  • Unstained, unfixed, and unoiled smears are preferred for cytologic examination. If submitting pre-stained smears, please do not oil or place coverslips on them (so they can be re-stained if necessary).
  • Submit all samples with a completed cytology request form, including relevant patient information, test requests and history.  See the Sample Submission page for further details.
  • Avoid exposure of any cytologic specimens to formalin. Cytologic samples should be submitted in a separate container from formalin-fixed samples (formalin leaches through capped or screw-top lids and affects staining quality as illustrated below.

Specimen exposed to formalin
Specimen exposed to formalin.

  • Do not allow cytologic specimens to come in direct contact with ice because cell lysis will occur (do not refrigerate smears).
  • Submit as soon as possible to the laboratory. This is particularly important with fluids, in which changes occur in the sample with storage, such as phagocytosis of erythrocytes (within a few hours) and bacteria (within 30 min).  This complicates result interpretation, so freshly made smears should ideally be provided along with any fluid samples (see below).

 

Tips on making cytology smearsSlide Steps A through C

For obtaining high quality smears from tissues or fluids, we recommend the following:

  • Use clean glass slides with frosted ends.
  • Place the aspirate near the frosted end such that the majority of the material is in the middle of the slide.  Our stainer cannot stain the edges or ends of the slides.
  • Make gentle squash smears (see images to right). 
    • Avoid making “splat” smears (spraying the sample on the slide without any kind of spreading). These are sub-optimal because they are very thick and the cells do not spread well.  This markedly hinders evaluation and interpretation.
    Thickly spread 'splat' smear
    Thickly spread ‘splat’ smear
    • Excessive pressure during smear preparation causes rupturing of cells and yields non-diagnostic smears. This can be a common problem with lymph node aspirates, since some lymphocytes are quite fragile and rupture readily.

    Excessive pressure applied during smear preparation

    Excessive pressure applied during smear preparation – all cells are ruptured.
    • Rapidly air-dry the slides.  Blowing on the back of the slide with a hair-dryer is best. This is very important because it optimizes cell spreading on the slide, allowing identification of individual cells and detection of small inclusions (e.g. bacteria) within the cells.

     

Tips on submission of fluid samples

For aspirates from body cavity fluids or lesions containing fluid, we recommend the following:

  • Submit fluid samples in EDTA (purple-top tube).
  • Submit some of the fluid in a red-top tube under the following circumstances:
    • If a need for culture is anticipated:  Use the ‘Other diagnostic tests requested’ field to the right of the Clinical Summary field on the cytology submission form to request a culture.
    • If any chemistry tests are to be performed on the fluid, e.g. urea nitrogen, creatinine, triglycerides, bilirubin.  Use the ‘Other diagnostic tests requested’ field to the right of the Clinical Summary field on the cytology submission form to request these additional tests.
    • If the fluid is very bloody. The presence or absence of clots in the red-top tube can help us distinguish whether the blood is from true hemorrhage or from collection-related blood contamination.
  • Store the fluid samples in the refrigerator and submit on ice packs (not in direct contact with the ice to prevent cell lysis).
  • To optimize results, also submit 2 or more smears of freshly collected fluid.
    • These smears should be made from unconcentrated fluid (called direct smears) or concentrated (centrifuged) fluid (called sediment smears). If making sediment smears, please do not use the entire sample (just centrifuge a portion of the sample). Please also indicate on the request form and on the smears (if making more than one type of smear), what type of smear has been prepared (direct or sediment).  Note that we can estimate the cellularity of the fluid from direct but not from concentrated smears.
    • Use the “wedge” or blood smear technique for making smears of fluids of non-mucoid samples (peritoneal, pericardial, pleural fluid). Use the “squash” technique illustrated above for mucoid or viscous fluids (tracheal wash, joint fluid, bile).
    • Rapidly air-dry the smears. This is critical for optimal results.
    • Indicate on the request form that smears have been submitted with the fluid (to ensure we are aware of their presence and make sure that we examine them, which is done at no extra charge).

     

Tips on submission of urine for cytology

  • Request a cytology smear exam on the cytology submission form and specify the method of collection (i.e. voided, cystocentesis or via catheter).
    • We recommend that a concurrent urinalysis is also requested on the sample for the best interpretation of the cytologic results. This does need to be requested as a separate test (Use the ‘Other diagnostic tests requested’ field to the right of the Clinical Summary field on the cytology submission form).
  • Submit urine in a red-top tube or sterile container.
  • Submit some of the urine in a separate red-top tube under the following circumstances:
    • If a need for culture is anticipated:  Use the ‘Other diagnostic tests requested’ field to the right of the Clinical Summary field on the cytology submission form to request a culture.
  • Store the urine in the refrigerator and submit on ice packs (not in direct contact with the ice to prevent cell lysis).
  • To optimize results, also submit 2 or more smears of freshly collected urine.
    • These smears should be made from centrifuged urine (called sediment smears). Please do not use the entire sample (just centrifuge a portion of the sample). 
    • Use the “line” (preferred) or “wedge” or blood smear technique for making smears of the urine sediment. See the diagram below for instructions on making line smears.
    • Rapidly air-dry the smears. This is critical for optimal results.
    • Indicate on the request form that smears have been submitted with the urine (to ensure we are aware of their presence and make sure that we examine them, which is done at no extra charge).

Line Smear Technique

Line smear technique 1 A. Place a drop of fluid near the frosted end of a slide and drag a spreader slide backward into the drop. B. The drop spreads along the junction of the two slides. C. Advance the spreader slide forward. D. When the spreader slide reaches no more than 2/3 the length of the slide, lift it directly upward.  This produces a line of concentrated cells at the end, instead of a feathered edge.  Briefly lift up the non-frosted end of the slide (marked *) to disperse the line towards the origin (this makes the line thinner and easier to examine) and rapidly air-dry the smear with a hair dryer (not shown).

1. This figure is duplicated with permission from: Diagnostic Cytology and Hematology of the Dog and Cat, Meinkoth JH, Cowell RL, Tyler RD, Morton RJ. Sample Collection and Preparation. p. 11, Copyright Elsevier (2008).

Special requirements for bone marrow samples

Please contact the lab if you would like advice on the collection and preparation of high quality bone marrow samples.  We require the following with each bone marrow aspirate submission:

  • 4-10 slides of freshly collected bone marrow are ideal. The more the better, so we can perform additional tests on the smears, as indicated.
    • Bone marrow aspirates should not be submitted as fluid samples (e.g. in EDTA). Marrow deteriorates very rapidly after collection and fluid samples are usually un-interpretable.
  • Recent hemogram results are essential for optimal bone marrow interpretation
    • Ideally submit a sample of peripheral blood in EDTA along with 2-3 freshly made peripheral blood smears for a hemogram along with a bone marrow cytology request.
    • Alternatively, provide a copy of recent hemogram results (within 24-48 hours of bone marrow collection), including a reticulocyte count if available. However, please note that we do prefer to examine peripheral blood ourselves with all bone marrow aspirate submissions as there have been many occasions on which we have detected something diagnostic in peripheral blood that was not specified in provided hemogram reports.