Download Through the Microscope: Practical Laboratory Skills Megan

Through the Microscope: Practical Laboratory Skills
Megan Brashear, BS, CVT, VTS (ECC)
DoveLewis Annual Conference Speaker Notes
Laboratory skills are vital for the veterinary technician. Interpreting red and white blood cells on
a hand differential, carefully looking for bacteria in a urine sediment, and evaluating effusion for
the presence of intracellular bacteria can all happen in a busy morning. By ensuring your
laboratory skills are top notch you can ensure better care for your patients and more revenue for
your hospital.
Performing a hand differential is part of a complete CBC along with a PCV/TS, platelet estimate,
and cell morphology exam. With modern machines able to reasonably differentiate cells, many
hospitals are bypassing the microscopic cell exam. Remember that machines are calibrated to
read the ideal and healthy scenario and may miss some subtle morphology changes, and in
emergency situations it is valuable for technicians to identify changes on a blood smear.
The first step to blood smear evaluation is to make a usable smear. Blood can be anticoagulated
(LTT) but remember that anticoagulant can dilute out platelet numbers so be sure there is
enough blood in the tube. If you are only able to obtain a small amount of blood, make the smear
as soon as possible. Even if using anticoagulated blood, ensure there are no clots in the tube.
Make sure the smear is not too thick, and contains a rounded, feathered edge. Allow the smear
to air dry, then perform a Wright’s Stain using care not to leave the smear in any stain for too
long which can make reading it more difficult.
Once the stained smear is dry, the hand differential should be performed with oil immersion
using 100x. Be sure that the area you are reading is a mono layer of cells and not out in the
feathered edge. Scan briefly to find the ideal place, and begin the differential.
When performing a hand differential, the first step is to identify and count the different types of
white blood cells (WBC). Scan through the smear until you have counted 100 WBC. The
following are types of WBC you can encounter:
 Segmented Neutrophils: most predominant WBC, phagocytic. Relatively clear cytoplasm
with a dark staining segmented nucleus.
 Band Cells: immature neutrophils that haven’t segmented yet. Tell of overwhelming
infection, inflammatory disease. Relatively clear cytoplasm with a dark staining U-shaped
nucleus (jump-rope). If in doubt between segmented or band cell, my money is on
segmented neutrophil.
 Lymphocytes: immune system cells, identify foreign substances and produce antibodies.
Small (slightly larger than red blood cells) with a dark staining round nucleus that takes
up almost the entire cell. If seen, cytoplasm is blue.
 Monocytes: circulate in the blood for a short time before entering tissues and becoming
macrophages. Largest WBC. Blue/purple abundant cytoplasm with a dark staining
‘globby’ nucleus. Often vacuoles in the cytoplasm.
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Eosinophils: degranulate during allergic reactions, help with parasitic response. Clear
cytoplasm with pink/red granules apparent with a dark staining segmented nucleus.
Basophils: rare, contain heparin and histamine. Clear cytoplasm with dark blue granules
often obstructing a dark staining segmented nucleus.
While counting WBC it is important to note the morphology of these cells and look for
abnormalities. Immature cells and toxic cells can point to disease processes. If too many WBC
look immature or toxic, it is a good idea to have the smear evaluated by a pathologist.
 Toxic neutrophils will have a lacy/moth-eaten appearance, and can be staged according
to other changes noted within the cell
o Toxic +1: Dohle bodies (small round dark inclusions in the cytoplasm and/or
nucleus)
o Toxic +2: Dohle bodies with cytoplasmic basophilia (small granules present in the
cytoplasm)
o Toxic +3: addition of granules evident in the nucleus
o Toxic +4: addition of vacuoles in in the cytoplasm
 Toxic lymphocytes will have a lacy/moth eaten nucleus (larger size does not
automatically mean toxic changes)
 Blasts are large, immature cells that are mostly nucleus and can look like lymphocytes,
but will be in much larger numbers. If these are noted, the slide should be sent to a
cytopathologist
While counting and studying WBC, don’t leave out the red blood cells (RBC)! Red blood cell
morphology should be examined and reported as well. When examining RBC pay attention to
cell size, cell shape, and cell color.
 SIZE: all RBC should be relatively close in size.
o Anisocytocis: cells of varying size. Report as mild, moderate or marked
 SHAPE: all RBC should be round with smooth edges
o Echinocytes: RBC with multiple small spiny projections from the edges
o Schistocytes: fragments of RBC
o Acanthocytes: RBC with a ‘jax’ like appearance (projections longer and less in
number than echinocytes)
o Spherocytes: smaller, perfectly round RBC (darker in color, lacking central pallor)
o Blister cell: RBC with a blister (either intact or ruptured)
 COLOR: all RBC should be relatively similar in color and stain uptake
o Polychromasia: cells of varying color. Report as mild, moderate or marked
 OTHER:
o Target cells: look like a target
o Heinz Body: looks like a round nose protruding from the RBC (fragment of nucleus)
o Howell-Jolly Body: single, small, dark round inclusion in RBC. Not clinically
significant
o Agglutination: if RBC are clumped together like a bunch of grapes
o Rouleaux: if RBC are stacked together like coins
Nucleated red blood cells can be seen when the bone marrow releases red blood cells early into
circulation. This can occur in cases of immune disease or regenerative anemia. Nucleated red
blood cells are often mistaken for lymphocytes, as they are of similar size and shape. In patients
where more than 7 nucleated red blood cells are counted per 100 white blood cells, a corrected
WBC count must be calculated. CBC machines often cannot differentiate nucleated red blood
cells from lymphocytes and this can create a false machine white blood cell count. To determine
the corrected white blood cell count:
Corrected WBC Count = [100/(nRBC +100)] x Machine WBC Count
Once the corrected white blood cell count is calculated it is the number that is used to
determine the absolute values of white blood cells in the differential.
Platelet estimates can be performed with every CBC and should also be utilized for any patient
with abnormal bleeding. When staining a blood smear for a platelet estimate, it is helpful to
leave the slide in the blue solution for 30 seconds to ensure a darker stain on the platelets. Begin
by scanning the edges of the slide and throughout the feathered edge for platelet clumps and
note their presence. If no clumps are present, a platelet estimate can be performed. Platelets are
smaller than RBC and stain blue (often lighter staining than the nucleus of WBC). To perform a
platelet estimate:
 Count the number of platelets in 10 oil fields
 Divide the total by 10 (average platelets/oil field)
 Multiply the average by 15,000
Performing a urine sediment exam is part of a complete urinalysis and can aid in the diagnosis of
diseases. Once the dipstick and specific gravity have been performed, spin the remaining urine
in the centrifuge, discard the supernatant, re-suspend the sediment, place a drop on the slide
and cover with a coverslip. Examine the sediment under 40x (no oil) and scan for cells, bacteria,
crystals, and casts
CELLS:
 Transitional Epithelial Cells: small amount is normal, large amount can signify transitional
cell carcinoma. Cells from the lining of the ureters, bladder and urethra, renal tubules,
etc.
 Squamous Epithelial Cells: small amount is normal, from the distal urethra and
prepuce/vagina. Can be seen in larger numbers from urine collected via catheter
 Red Blood Cells: small numbers often seen, <5/HPF normal
 White Blood Cells: <5/HPF normal, larger than RBC, more grainy
CASTS:
 Originate from distal tubules and collecting ducts of kidneys, usually indicative of renal
disease. Will have a definitive border, oblong
 Coarse granular
 Fine granular
 Cellular
 Waxy
 Hyaline
CRYSTALS:
 Influenced by diet, pH, concentration of urine, temperature
 Struvite – rectangular, coffin shaped
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Calcium oxalate dihadrate – perfect squares, X in the center
Bilirubin – needle like bursts
Other, rarely seen: cysteine, ammonium biurate, calcium carbonate
BACTERIA:
 Cocci – not to be confused with fat droplets, small and round
 Rod
AMOURPHOUS DEBRIS
Report findings as <5, 5-10, 10-20, 20-50, TNTC /HPF
Technicians must be able to identify intracellular bacteria in cavitary effusions and urine.
Preparation of the sample involves centrifuging the sample (the urine setting is appropriate) and
discarding the supernatant. Using the sediment, place a drop onto the slide and make a smear
similar to a blood smear. Stain in the Diff Quick as you would a peripheral blood smear. Observe
the sample under oil immersion and look for evidence of both extra cellular and intracellular
bacteria. Identify if the bacteria is cocci, rods, or both. Some technicians may choose to not
centrifuge the sample prior to creating the smear; if this is case ensure that the same method is
used throughout for a single patient.
Performing a saline agglutination test is a quick test to determine red blood cell agglutination in
cases of immune mediated disease.
 Using anti-coagulated blood, apply a drop to a microscope slide
 Add to the blood one or two drops of 0.9% NaCl
 Tilt the slide to mix and observe for clumping (will appear like grains of sand) within the
sample. If clumping is evident this could be rouleaux, the sample still must be examined
under the microscope
 Observe the sample under 10x power and looking towards the edges where the cells are
in a single layer, look for agglutination (red blood cells clumping together like clumps of
grapes). If the cells are stacked like coins, this is rouleaux. Adding more saline to the
sample should break up rouleaux and help differentiate agglutination
 Test results are positive or negative