Hematology Lab Slideset One Flashcards
Different sizes of purple top tubes – 7ml, 3ml, 2ml, and 0.5 ml volumes. EDTA is the anticoagulant of choice for hematology specimens. Better preservation for counts and good cell morphology. Always choose the correct size tube for the sample volume.
EDTA – Tube with small amount of blood – EDTA induced artifacts will occur if tube is not properly filled. Should be at least half-full. Improper filling will:
- Falsely decrease the PCV due to shrinkage of RBC
- Falsely increases the plasma protein reading on the refractometer.
Making blood smear – small drop (2-3 mm diameter) of well mixed blood on one end of clean slide. Smears should be made with fresh blood.
Pusher slide – Place the end of a second slide (“spreader”) against the surface of the first slide, holding it at a 30-45 degree angle.
Blood spreading – as the spreader slide makes contact with the blood, the blood will spread along the edge of the spreader slide. Push the spreader slide forward with a steady, even motion making a thin blood film.
Pushing blood across – the thickness of the blood smear should decrease from beginning to the feathered edge. The thickness depends on the size of the drop of blood, the angle of the spreader slide, and the speed with which the spreader slide is pushed.
Finished slide – quickly air-dry or heat-fix smear to prevent artifacts. If smear is not going to be stained soon, it’s a good idea to turn the smears over so that roaches cannot dine on the blood.
5 problems unstained smears –
- “picket fence” – hesitation while pushing the blood across the slide
- “Holey” – greasy or dirty slide
- “Half slide” – not letting blood spread entirely across the edge of the spreader
slide
- “Short slide” – angle of the spreader was too high
Blue Foil – Romanovsky Stains
Diff-Quik coplin jars – Diff-Quik stain is a modification of the Wright’s stain technique which allows you to stain a slide in 15 seconds. Smear is fixed in a methanol fixative solution.
- 5 one second dips
- Solution I – stains the eosinophilic (red) cellular components; 5-6 one second dips
- Solution II – stains the basophilic (blue) cellular components; 5-7 one second dips
- Coplin jars should be tightly covered when not in use
Stained finished slides
Artifact-stain precipitate – not commonly seen with Diff-Quik stain. More common with automated stainers.
Artifact – water damage – in focus – result of slow drying
Artifact – water damage – out of focus – can be mistaken for RBC abnormalities
Artifact – Hemolyzed RBC’s – result of red cells lysing from exposure to excessive humidity. This will appear in smears that are refrigerated before staining
Blue Foil – Microhematocrit procedure
PCV – filing the capillary tube for PCV
Sealing the end of the capillary tube before spinning.
Placing capillary tube in microhematocrit centrifuge. Length of spinning dependent on the centrifuge and species. However, a 5 minute spin is generally recommended.
PCV reader card – packed cell volume measures the volume of red blood cells expressed as a percentage of the volume of whole blood in a sample. Care should be taken that the top of the plasma column should be aligned with the 100% line and the bottom of the packed RBCs should be on the 0% line.
Normal vs. anemic PCV – Normal plasma appearance
Hemolyzed plasma – will increase TPP on refractometer.
Lipemic plasma – will obscure TPP on refractometer.
Icteric plasma
Standard vs. Veterinary Refractometers for Total Plasma Protein (TPP) determinations.
Scale of standard refractometer – scale for protein, urine specific gravity, and refractive index.
Scale of Veterinary refractometer – protein scale and 2 specific gravity scales. One for cats and one for other. Reading for specific gravity goes up to 1.060.
Blue Foil – Electronic Cell Counter
Automated cell counters utilize a suspension of cells passing through an intensified zone of electrical current or light. As individual cells pass through the sensing zone, they cause a rapid sequence of pulses, which are amplified, measured and counted. For leukocyte counting, any cell with a nucleus is counted. (including avian RBC’s, nucleated RBC’s.
The QBC method utilizes a fluorescent dye and density gradient cell layering within the buffy coat to measure the separated packed volumes of red cells, white cells, and platelets. A plastic float inserted in a specially coated glass capillary tube settles within the buffy coat during centrifugation, and expands the leukocyte and platelet layers. A measurement is taken of each layer using the QBC instrument and results are calculated. Nucleated RBC’s are not counted with this method.
Blue Foil – Hemacytometer Manual method
Unopette system – diluting the sample is the first step in a manual leukocyte count and this system is the method of choice because of its accuracy, precision, speed, and simplicity.
-Reservoirs contain a premeasured amount of diluent and also consists of a uniform capillary tube for dispensing the sample amount. ALWAYS read the directions!!! Unopets have several different kits, which require certain counting procedures and calculations.
Grid from Hemacytometer – a hemacytometer or counting chamber is a device used for counting the number of white blood cells in a certain area. A small amount of diluted blood is deposited uniformly over the chamber. The grid is divided into 9 large squares. The 4 corner squares are further divided into 16 squares and the center is divided into 25 squares. Each of the 9 squares is 1mm in length and 1 mm in width with the coverslip creating a depth of 0.1mm. The entire 9 squares equal 0.9 microliter in volume.
-Hemacytometers should not be scratched and coverslips should be optically plane hemacytometer coverslips to accurately control depth. Care should be taken to avoid underfilling or overfilling of the chamber. This will change the chamber volume and result in an inaccurate count. Should be read using the 10x objective and minimum amount of light.
Blue Foil – Standard Hemacytometer Formula – counts are based on a volume of 1 micro liter. This formula will bring this count to this volume depending on the number of squares counted and the dilution factor.
Platelet clumps – 10x objective (100 magnification) – Always start the blood smear exam by looking for obvious clumps. An accurate platelet count cannot be obtained if clumped. Cats usually clump!!
- WBC in feathered edge – look for a typical WBC distribution on the feathered edge as well as in the counting area. There will be a certain number of cells that will go to the outer edge and no WBC’s will be in the remaining area of the smear. This due to poor blood smearing technique.
WBC clumps – WBC clumps occurring in any area of the smear will result in an inaccurate total WBC count. Clumps will decrease any count obtained (If a count is obtainable)
Microfiliria – Blood parasite found mainly in canine. Will observe on low power.
Bad area of slide for differential – TOO thick.
Good area of the slide for differential – nice monolayer of RBC’s and WBC’s are not distorted.
Neutrophilis (“seg”) – most common mature sell in peripheral blood. The nucleus is stained dark purple especially where there is chromatin clumping. The outline of the nucleus is lumpy and may be twisted or coiled. May be segmented or simply pinched in places. The cytoplasm will appear a faint pink and you may see small pink granules. Very similar in all species.
Lymphocyte and Neutrophil – second most common cell seen in peripheral blood. Lymphs can vary in size. The nucleus is round or slightly indented. The chromatin is heavily clumped and stains a deep purple. Cytoplasm, when visible, stains a light to medium purple that is similar in all species except bovine. Bovine tends to have more bizarre lymphs.
Reactive lymph – Cytoplasm stains an intense blue. These lymphs have been antigenically stimulated to produce antibodies. Note a few, moderate, many seen. Note the less intense staining cytoplasm adjacent to the nucleus known as a perinuclear halo.
Monocytes – the largest mature white cell in peripheral blood. The nucleus shape can be round, indented, lobed, or ameboid (pleomorphic). Chromatin material is fairly even dispersed and the outlined is smooth. Cytoplasm is slightly blue gray and may appear more granular giving a “ground glass” appearance vacuoles are frequently present on the cytoplasm. Similar in all species.
Eosinophils – granules stain red. Nuclear structure is same as neutrophil. Increased with parasitic infections and allergy.
-Canine-granules are round, may vary in size even within the same cell. May be a single granule the size of a red cell or packed with tiny ones.
- May be mixed.
- Feline – Granules are red, rod-shaped, not-refractile, and numerous
- Equine – Granules are very large, tightly packed and completely fill the cell
(raspberry-like appearance)
- Bovine – Granules are very small and pack the cell
Equine Basophil – granules are purple, irregular in size and shape, and may be numerous or few in number. Most of the time, the granules may obscure the cytoplasm nucleus.
Canine Basophil and Neutrophil – Basophils are very rare in peripheral blood. Nucleus varies from sausage-shaped to highly segmented. The basophilic granules decolorize almost as easily as they stain and you may see only a few faintly purple or black ones. They can look like a “dirty” monocyte.
Feline Basophil and Neutrophil – Basophils are rarely seen in cats. The cell has lightly gray cytoplasm filled with small, round, lavender granules. May be very pale and may be mistaken for a dull staining neutrophil.
Neutrophils and Band (immature neutrophil) – The band neutrophil is present in small numbers in health. The nucleus is horseshoe shaped with parallel sides The chromatin is less heavily clumped and nuclear outline is smooth rather than lumpy.
Neutrophil, bands, metamyelocyte – In bacterial – induced inflammation states, the need for Neutrophils is great to fight infection. The bone marrow may begin to throw out immature neutrophilic forms, creating a “left shift”.
Blue Foil – Toxic changes – Dohle bodies, vacuolation and basophilic cytoplasm. Toxic states result in changes in the nucleus and/or cytoplasm of the neutrophil due to interference in maturation.
Dohle bodies – bluish cytoplasmic inclusions. Can occur very commonly in cats.
Diffuse Basophilia (Basophilic cytoplasm) – blue staining cytoplasm in neutrophils, which are normally colorless.
Vacuolization (foaminess) of cytoplasm.
Left shift with bands and metas – Note the Dohle bodies, vacuolation and basophilic cytoplasm.
Pyknotic cell – Disintegrated nucleus in leukocyte; cannot differentiate which type. Main reason for this is old age of blood sample. Always make fresh smears when submitting CBC’s through mail or by courier.
Blue Foil – Erythrocyte morphology – Red cell evaluation is the next step in the blood smear exam. It is important to note any of these changes.
Canine RBC – nice central pallor, occasional target cells, occasional polychromasia, slight anisocytosis.
Blue Foil – Regenerative anemia – red cell morphology is most helpful in diagnosing causes of anemia. Increased anisocystosis.
Anisocystosis and Polychromasia – These are due to the release of immature red cells from the bone marrow in effort to replace lost blood. These immature cells are larger than the mature cells and it is the mixture of large and normal cells that create the picture of increased cell size variation (aniso). Polychromasia refers to the way the immature (larger) red cells stain with Wright’s stain. Due to remnants of RNA, these RBC’s take on a bluish tinge as well as the reddish tint (from hemoglobin) of mature cells (polychromatophilic). This is an indication of bone marrow regeneration.
Reticulocyte Count – Erythrocytes with deep purple or black inclusions caused by NMB stain are reticulocytes. Count 1000 RBC’s and give a percentage of retics. In cats, there are two basic types of retics, making a count more difficult. The aggregate type is the large, intensely stained granules and the punctuate type is very fine, diffuse, scattered granules. Counts in cats should include only the aggregate form; may comment on the presence of punctuate form (few, moderate or many seen).
NRBC’s – Earlier RBC’s than polychromatophilic cells. When blood loss if very severe or marrow is damaged, the bone marrow can release these early precursors, may be confused with lymphocytes but these have more cytoplasm and a small darker staining nucleus.
Blue Foil – Correction for NRBC’s – If more than 10 NRBC’s are present per 100 WBC’s then total WBC count must be corrected.
Howell Jolly body – nuclear fragments. Remainder of incomplete nuclear extrusion from the red cell. These bodies are dark purple bodies occurring singly or in multiples. An occasional one is normal but an increase may be seen in a regenerative anemia.
Basophilic Stippling – presence of blue granules scattered throughout the red cell in a Wright-Stained smear. These granules are RNA that stains with Wright’s stain.
Poikilocystosis – Variation in shape – may be valuable information in some disease states but may only be artifacts. Crenation is a form of poik. That is not of importance in the majority of cases and is mainly an artifact from the smear making technique. Crenated RBC’s have several blunt of pointed evenly spaced surface projections.
Target Cells – RBC’s with increased diameter and decreased cell thickness. These cells have a well-defined central area of pigmented material surrounded by a clear zone with no pigment and a dense ring of cytoplasm about the periphery of the cell. These cells are thin and they tend to fold or become bowl-shaped. Their presence is of no diagnostic importance.
Schistocyte and spherocyte – schistocytes are irregular red cell fragments resulting from trauma in the circulation. Blood may contain a rare schistocyte, but an increase is significant. Seen in DIC. Spherocytes are red cells that have a decreased cell diameter and a normal amount of hemoglobin. Their appearance in blood can indicate immune-mediated hemolytic anemia.
Acanthocytes – red cell with several unevenly distributed surface projections that are irregular in length, thickness, and distribution. The projections have knobby ends. May mean hemangiosarcoma or liver disease in canine. May see in bovine normally.
Heinz bodies with Wright’s stain – Heinz bodies are denatured hemoglobin, irregular in shape, and since they don’t take up stain with Wright’s stain, they appear as a single bleb on the margin of the red cell.
Heinz bodies with New Methylene Blue – Heinz bodies are easier to see by using a supervital stain. With NMB, these bodies appear as single large black granules. An increase may indicate hemolytic anemia associated with toxic drugs or plants.
Haemobartonella felis – parasite seen on red cells of cats, usually with a regenerative anemia.
Anaplasma marginale – red cell parasite of bovine red cells.
