Laboratory Methods in Hematology

Blood Collection

Proper blood collection or handling is critical; improper technique can result in inaccurate blood cell counts and morphologic artifacts.

> Sample quality is the major contributor to analytical errors.

Hematological assessment requires liquid blood.

> Blood should be drawn into vials or syringes that contain anticoagulant. EDTA is the anticoagulant of choice for most hematology.

• Smears should be prepared as soon after collection as possible; prolonged exposure to EDTA produces artifacts in neutrophils and platelets.
• Red cells show increased susceptibility to lysis after 24 hours in EDTA.

> Sodium citrate is recommended for platelet and coagulation studies.

Figure 3-1. Collection materials for hematological assessment include syringe, needles, and purple topped collection tubes that contain EDTA. Blood in tubes should be mixed by inversion rather than shaking.

Figure 3-2. EDTA tubes should be filled to their labeled capacity. Short filled tube (left) has an excessive amount of EDTA relative to the blood volume. Short filled tubes cause a false increase in total plasma protein content and a false decrease in PCV and RBC count. Shrinkage of RBCs causes a reduction in the MCV.

> Heparin should not be used to collect blood for canine and feline hemogram interpretation. It fails to prevent platelet aggregation and causes morphologic changes in white cells.

Self-drawing evacuated tubes (e.g., Vacutainer®, Becton Dickinson, Franklin Lakes, NJ or Monoject®, Kendall, Mansfield, MA) are preferred to syringes.

> They should be allowed to fill to capacity to achieve the appropriate blood/anticoagulant ratio (see Fig. 3-2).

> Invert the vial several times after filling to ensure thorough mixing.

Rapid aspiration or transfer of blood with a syringe through a small gauge needle can cause cell lysis.

Handling the Sample

Blood should be processed as soon as possible after collection.

> Blood films should be made immediately.

> If a delay is anticipated before processing further, the blood should be refrigerated.

> Excessive time at room temperature can cause autolysis.

> Platelet counts are most affected by delays in processing. Platelets have short life spans and tend to clump over time, even in the presence of anticoagulants. Platelet counts performed more than 4 - 6 hours after collection are suspect (See Fig. 4-23).

Blood samples should be mixed again several times immediately before a portion is removed for testing; avoid prolonged mixing to prevent physical trauma to cells.

Blood Films

Well-prepared blood films are prerequisite to accurate assessment of the hemogram.

Use only new, clean slides.

Place a small drop of blood near the frosted end of one slide (Fig. 3-3a).

> Place another slide at an angle of about 30 degrees to the first; draw back until it touches the drop of blood in the acute angle between the slides (Fig. 3-3b and Fig. 3-3c).

> After the blood has spread to within 2 - 3 mm of the edge, push the second slide quickly and smoothly across the full-length of the first (Fig. 3-3C).

Figure 3-3a. Preparation of a blood film.

Figure 3-3b. Small drop of blood is placed on a glass slide.

Figure 3-3c. A second slide is used to spread the blood. Lowering the spreader slide produces a longer blood smear. Raising the spreader slide produces a shorter smear.

> A well-formed smear has a flame shape.

> Prepare several slides from each patient.

An alternative method preferred by some hematologists is a variant of the coverslip method.

> Using a PCV tube, place a drop of blood slightly off center on a CLEANED (IMPORTANT) glass slide.

> Place a second cleaned glass slide on top and watch the spread of blood by capillary action between the two slides.

> Just as this stops, slip the upper slide off (DO NOT LIFT OFF!). This most often results in two thumbprint-sized perfect smears.

> The monolayered areas are the entire center of these smears with a thicker but small perimeter.

Air dry the smears quickly and store at room temperature until processed.

> Do not blot or wipe dry; this introduces scratches.

> Do not refrigerate; the condensation that forms on cold slides can lyse cells.

> Keep away from formalin.

> Do not fix until ready to stain, but keep covered; flies will consume blood on air dried smears.

Staining

Romanowsky stains (Wright, Giemsa, and modified quick stains) afford the best overall morphologic assessment of the hemogram.

> These stains contain both an acid stain (usually eosin) and a basic stain (such as methylene blue).

> Structures rich in basic compounds, such as eosinophil granules, bind the acidic dye and are stained red. Acidic structures, such as DNA/RNA or basophil granules, are stained blue by the basic stain.

New methylene blue

> A supravital dye used for reticulocyte counts and to accentuate Heinz bodies.

> Mix a few drop blood with 1 - 2 times as much of 0.5% new methylene blue in physiologic saline, allow to stand a few minutes and use to make blood films.

Common Artifacts/Issues

Stain precipitate

> Stain that is old or has been left open may deposit precipitate on the slide that can be mistaken for hemoparasites.

> Keep stain fresh and always covered when not in use. Periodically filter or replace to minimize precipitate.

Over- or under-staining

> It is often necessary to experiment with staining procedures to avoid over-staining or under-staining.

> In over-stained slides, all cells are deeply colored. The red cells appear to be more dense and more basophilic (blue) than normal. Over-staining can obscure important cell details (Fig. 3-4).

> In under-stained slides, all cells are pale. Cellular details of the leukocytes are barely distinguishable and red cells are very faint. This should not be confused with hypochromia.

Figure 3-4. Blood film that has been overstained with Wright Giemsa. The RBCs are a dark blue grey color that makes the identification of polychromasia impossible. The neutrophil on the right contains a canine distemper inclusion in the cytoplasm (arrow).

Sample Evaluation

Morphological Assessment

Microscopic examination of a blood smear is an essential part of any hematological evaluation, regardless of the method used to enumerate the cells.

> Blood cell counts alone are not sufficient to adequately evaluate the hemogram.

> The size, nature, and condition of cells and platelets provide information vital to characterize disease processes.

• Some diseases, for example, blood parasites and certain neoplasms, can be diagnosed directly from examination of the blood film.

A systematic approach to evaluation of the blood smear is essential to obtain accurate and complete results.

> A common error is to begin to identify and count the white cells immediately at high magnification, failing to observe the characteristics of the leukocytes, erythrocytes and platelets.

> Scan at low magnification (10 - 20X) for rouleaux formation and for RBC, WBC or platelet aggregation, which can cause erroneous cell counts in most automated counters.

• Estimate the total number of leukocytes (Fig. 3-5a, Fig. 3-5b and Fig. 3-5c) and develop a mental image of the appearance of typical leukocytes of each cell line (neutrophil, eosinophil, lymphocyte, monocyte) (Fig. 3-6).
• Evaluate the red cells for evidence of polychromasia, anisocytosis, hypochromasia, poikilocytosis, etc.
• Note any unusual findings (atypical cells, parasites).

Figure 3-5a. Series of blood smears with increasing WBC counts. Blood film from a dog that is severely leukopenic and anemic. The density of RBCs and WBCs is markedly reduced.

Figure 3-5b. Series of blood smears with increasing WBC counts. Blood film from a dog with normal WBC count.

Figure 3-5c. Series of blood smears with increasing WBC counts. A marked leukocytosis (WBC=158,000/uL) is evident.

Figure 3-6. Major abnormalities can be detected by scanning a smear. The majority of leukocytes in a normal canine or feline blood film should be segmented neutrophils. In this smear, most of the WBCs are large mononuclear cells. Note that these cells are larger than a neutrophil (arrow) and are most likely atypical lymphocytes or neoplastic cells.

> Oil immersion magnification.

• Examine erythrocytes and confirm observations made at low magnification (size, shape, color, abnormalities and any inclusions).
• Examine platelet morphology and distribution; estimate relative number.
• Examine leukocyte morphology (abnormalities and inclusions).
• Perform differential leukocyte count if not using automated equipment.
• If using automated equipment, estimate the differential count and compare to that reported by the instrument; this serves as an in-clinic quality control check!

Quantitative Methods

Packed Cell Volume (microhematocrit)

> Microhematocrits are accurate and repeatable. Instrumentation and supplies are inexpensive and suitable for all practices (Fig. 3-7aand Fig. 3-7b).

> High speed centrifugation is used to separate cells from plasma.

> The major source of error is trying to save time by not allowing the sample to spin the full amount of time. This produces an overestimate of the PCV because the plasma and cells are not fully separated.

> Hematocrits may also be computed and reported by in-clinic automated analyzers.

> The appearance of plasma in hematocrit tubes also can provide important information. Icterus, hemolysis, and lipemia may all be detected (Fig. 3-8).

Figure 3-7a. Microhematocrit centrifuge to measure PCV. The microhematocrit centrifuge produces accurate measurements of circulating RBC mass and provides an opportunity to assess abnormalities in plasma color and to measure the total protein concentration by refractometry.

Figure 3-7b. Card reader to measure PCV. The microhematocrit centrifuge produces accurate measurements of circulating RBC mass and provides an opportunity to assess abnormalities in plasma color and to measure the total protein concentration by refractometry.

Figure 3-8a. Abnormal plasma colors in canine plasmas. In the large blood tubes, from left to right, icterus, normal plasma, lipemia with hemolysis, and hemolysis are noted.

Figure 3-8b. Abnormal plasma colors in microhematocrit tubes are more difficult to observe because of their small diameters. Icterus, lipemia, hemolysis, and normal plasma are displayed left to right. The first two samples are animals that were anemic. Note the markedly reduced PCV.

Total Plasma Protein

> Total plasma protein can be measured easily by refractometry (Fig. 3-9). For dogs and cats, the reference range is generally between 5.5 g/dl and 7.5 g/dl.

> Low total protein values reflect one of the following abnormalities

• Protein losing nephropathy (characterized by proteinuria).
• Protein losing enteropathy (usually associated with chronic weight loss and diarrhea).
• Loss of lymph (check for pleural or peritoneal effusion).
• Chronic or severe blood loss (check hematocrit!).
• Lack of protein production by the liver.

> Elevated protein values reflect either hemoconcentration or increased globulin production.

• Hemoconcentration can cause elevations in red cell parameters, concentrated urine specific gravity, and elevations in serum electrolytes.
• Increased globulin production is most commonly associated with inflammation; an inflammatory leukogram is generally present.

> Evaluation of serum protein and serum albumin levels are useful in further clarifying the interpretation of plasma protein abnormalities.

Figure 3-9. Hand held refractometers can be easily used to determine total plasma protein as well as urine specific gravity.

Hemoglobin Concentration

> Hand held hemoglobinometers provide a simple and rapid method to estimate hemoglobin concentration if automated equipment is not available.

> Some in-clinic chemistry analyzers measure hemoglobin in whole blood samples electrochemically.

Manual Cell Counts (Fig. 3-10a and Fig. 3-10b)

> Microscopic counting of red and white cells is the oldest and most time-consuming method of determining blood cell counts.

> Microscope and special slide (hemocytometer) are required.

• The hemocytometer is calibrated to hold a known volume of fluid between the cover slip and a grid etched on the slide
• Can also used for counting cells in other body fluids and effusions.

> Dilution of blood

• Different dilutions for red and white cell counts are necessary.
• For white cell counts, erythrocytes are lysed.
• The Unopette® System (Becton Dickinson, Franklin Lakes, NJ) provides a convenient and reproducible system of pre-measured diluents (Fig. 3-11).

> Red cell count

• Red cells can be counted using a hemocytometer, but the margin of error is high even among skilled medical technologists.
• Red cell counts themselves offer little additional information over the hematocrit. They are needed to calculate of red cell indices, but manual counts are generally too variable to yield reliable information.

> Reticulocyte count

• Performed by counting at least 1,000 erythrocytes on a smear made with supravital (new methylene blue) stained blood.
• The absolute reticulocyte count is determined by multiplying the red cell count times the percent of reticulocytes.

> Differential white cell count

• The stained smear should be examined with the oil immersion lens.
• 100 - 200 white cells are characterized by type. The larger the number counted, the smaller the margin of error.

• Avoid areas where cells are overlapping or distorted.
• Use a consistent method to scan the slide to ensure random sampling and avoid counting the same area twice.

• The percent of each cell type is multiplied by the total white cell count to determine the absolute number of each leukocyte/ul.

• Absolute numbers (not %) should always be used in evaluating the hemogram.

Figure 3-10a. Hemocytometer grid lines. Erythrocyte and leukocyte count. Red = zones to be counted under high power for erythrocytes. White = zones to be counted under low power for leukocytes.

Figure 3-10b. Left: The hemocytometer is designed to keep the cover glass 0.1 mm above the grid so that there is a known volume of fluid over each grid area. Right: The arrow indicates the direction in which the count should be made. Triple ruling: Cells touching top and left center lines are counted (shaded cells for the first row). Cells touching bottom and right center lines are not counted. Double ruling: Cells touching top and left outer lines are counted. Cells touching bottom and right inner lines are not counted. Reprinted with permission from Benjamin MM, Outline of Veterinary Clinical Pathology, 3 Ed., Ames, IA, the Iowa State University Press, 1978. Available from amazon.com.

Figure 3-11. Unopette® diluting pipette and reservoir for counting WBCs and platelets in a hemocytometer.

> Platelet count

• Can be performed using hemocytometer and ammonium oxalate diluent but it is difficult to achieve accuracy even among skilled medical technologists.
• Platelet count can be estimated from the blood smear.

• 10 - 12 platelets per oil immersion field (100x nose piece objective) is an appropriate number in the dog and cat.
• In the absence of obvious clumping, fewer than 10 - 12 platelets per 100x field suggests thrombocytopenia and indicates the need for a quantitative platelet count.

Advantages

> Manual counting is the least expensive in terms of equipment, supplies.

> Can be performed in mobile clinics.

Limitations

> Time consuming; most expensive in terms of professional staff time.

> Greatest variability/unreliability in results.

• Inherent error is 20% or more, even among experienced technologists.
• Requires a high level of care and skill to produce accurate and precise results.

Automated Cell Counters

> Several kinds and brands of electronic cell counters are now available for use in veterinary hospitals.

> All produce more accurate counts than manual methods and require less technician time.

• Compared to manual methods, a much larger number of cells are counted (several thousand) producing repeatable differentials and absolute counts.

> Some automated in-office equipment will produce partial or, more recently, complete differential counts.

• The most recently introduced systems also produce accurate platelet and reticulocyte counts.

> In-office equipment offers the advantage of immediacy, producing results during the visit.

• It is therefore especially well suited to acute care and to pre- anesthetic and well-patient screening.

> Systems are of two types: semi-automated and fully automated.

• Semi-automated equipment requires sample preparation (for example, dilution) by a technician; fully automated equipment performs all steps subsequent to obtaining the sample itself.

> All automated equipment must be properly maintained and periodically re-calibrated to produce accurate and consistent results.

• Written standard operating procedures should be available and understood by all persons who will be operating the equipment.
• The manufacturer's recommendations for instrument calibration, quality control, and maintenance should be followed closely. Establishing guidelines for in-clinic quality control is essential.
• Instruments designed for analysis of human blood samples require modification and validation to produce accurate hemograms for other species.

> Each method has its strengths and limitations (see below); like all techniques in medicine, automated cell counting must be used intelligently.

• The clinician should first rule out laboratory or collection artifact when the lab data are incongruent with the clinical assessment.

Impedance (Fig. 3-12)

> All impedance counters make use of Coulter principle.

> A dilute solution of cells in electrolyte solution is drawn through a small aperture between two electrodes.

> When a particle passes through the opening, it causes a change in electrical impedance and a measurable voltage pulse.

• The magnitude of the voltage change is proportional to the size of the cell.
• Voltage pulses are detected, analyzed and counted electronically.

> The red cell count actually includes both red and white cells, but because the proportion of white cells is usually small, the effect is insignificant unless the animal is simultaneously anemic and leukemic.

> Red cells are lysed in order to obtain a white cell count.

> Erythrocytes and platelets are differentiated on the basis of size (magnitude of the voltage change).

Figure 3-12. Impedance counters rely on the Coulter principle. When a particle passes through a narrow orifice, it causes a change in the resistance. The magnitude of the change (corresponding to cell size) and the number (corresponding to the number of cells) are recorded by the detection circuit.

Advantages

• Faster and more effective use of staff time than manual methods.
• Newer models store calibration settings for different species.
• Fully automated impedance counters are available for veterinary use that simultaneously perform RBC, WBC and platelet counts, determine mean corpuscular volume, and calculate hematocrit, MCHC and MCH.
• Some of the newer systems produce three-part differentials.
• Relatively inexpensive to operate and use.

Limitations

• Major limitation is the lack of any reticulocyte data.
• Major limitation is the inability to produce a complete differential count. Impedance counters group granulocytes into one category.
• Older models still require multiple step sample processing (dilution); red and white cells must be counted in separate steps.
• Relatively poor ability to differentiate among white cells and to differentiate white cells from nucleated RBCs.

• WBC cell count must be corrected for nucleated RBCs.

• The overlap in size between feline platelets and erythrocytes leads to overestimates of the erythrocyte count and underestimates platelet numbers.
• Clumping of leukocytes leads to undercounting, and artifactual leukopenia.
• Only nuclear material is analyzed not cytoplasmic.
• Does not distinguish between bands and separated neutrophils; consequently a left shift cannot be determined from impedance data.
• Fibrin strands resulting from microclotting can plug the aperture and cause falsely decreased counts.
• Aggregates of platelets may be counted as white cells; this is especially problematic in cats.
• White cell counts are determined after lysing red cells; failure of RBCs to lyse completely produces falsely elevated white cell counts.

• Erythrocytes with Heinz bodies don't lyse; hence, cats with large numbers of Heinz bodies may have falsely elevated WBC counts, hemoglobin measurement and the RBC indices (MCH, MCHC).
• Polychromatic erythrocytes are more resistant to lysing and lead to falsely elevated WBC counts.

Quantitative Buffy Coat (QBC) Analysis (Fig. 3-13)

> Based on differential centrifugation

• Under high speed centrifugation, blood separates into plasma, buffy coat and red cells.
• The buffy coat itself is divided into layers based the relative density of the white cells and platelets; the platelets are the least dense and form a layer just beneath the plasma. Followed by monocytes and lymphocytes the granulocytes are the most dense of the white cells and come to rest immediately on top of the erythrocytes.

> Uses special acridine orange-coated tubes that include a cylindrical float with the same density as the buffy coat. When a tube is centrifuged, the float effectively reduces the diameter of the tube in the region of the buffy coat, causing it to spread along a greater length of the tube, enabling resolution of the various layers.

> The acridine orange stains nucleoproteins and other cellular components, which fluoresce when exposed to ultraviolet light.

> An automated reader (QBC® Vet Autoread, IDEXX Laboratories, Westbrook, Maine) scans the buffy coat layer and records the intensity of fluorescence produced by DNA and RNA/lipoproteins. Abrupt changes in slope are used to detect changes in cell type. The relative width of each band of fluorescence is used to calculate the population of cells.

• The results and a graph of the pattern are printed out. The software is programmed to alert the operator of unexpected or uninterpretable results or patterns.

Figure 3-13. The quantitative buffy coat (QBC) uses centrifugation to separate cells by density. A "float" in the QBC tube reduces the volume of the lumen in the area of the buffy coat, causing the cells to be spread out over a greater distance. Cells types are differentiated by fluorescence in the presence of acridine orange.

Advantages

• The QBC is an efficient and economical way to screen blood samples.
• Instrument is simple to operate and relatively fast, producing results in about 7 minutes making it especially valuable for pre-anesthetic, acute care and in-office screening procedures.
• Hematocrit, hemoglobin, and white cell and platelet counts correlate well with reference methods.
• System flags abnormal or unexpected result.

Limitations

• The major limitation is the inability to produce a complete differential count. QBC cannot distinguish between lymphocytes and monocytes. Canine eosinophils inconsistently separated from lymphocytes and monocytes.
• Does not distinguish between bands and segmented neutrophils. The degree of left shift cannot be determined from the QBC data.
• Tends to underestimate frequency of leukopenia
• Algorithms used to calculate RBC and WBC counts assume normal cell size and structure. They are thus invalidated by conditions such as microcytosis, hypochromasia, and cell immaturity. Assessment of a stained blood smear is essential to rule out conditions that could lead to inaccurate differential counts.

Flow Cytometry (Fig. 3-14)

> Laser flow cytometry is the newest and most accurate method of automated cell counting.

> Generally considered the gold standard in automated hematology

• Used in most commercial laboratories, but until recently was cost-prohibitive for in-office use.

> A suspension of blood cells is broken into micro-droplets and passed through a laser beam.

> Cells absorb and scatter the laser light. Each cell type produces a characteristic "signature" depending on its size, nuclear configuration and cytoplasmic inclusions.

> The most sophisticated models generate a five-part differential, platelet count, reticulocyte count, and red cell indices.

> Fully automated models require no dilution or other sample preparation.

> Advanced units include particle standards in the diluent.

Figure 3-14. Laser flow cytometry detects and counts individual cells in micro- droplets as they pass through a laser beam. Each cell type scatters the laser in a characteristic "signature" based on its size, nucleus and cytoplasmic contents.

Advantages

• Because laser flow cytometry systems evaluate multiple parameters (nuclear and cytoplasmic material) of each cell or platelet, they produce more accurate and reliable counts than other methods.
• Clumped cells or platelets can be detected and ignored.
• Large platelets (frequently encountered in cats) can be distinguished from erythrocytes due to difference in light scattering caused by the platelet granules.
• Flow cytometers are able to rapidly count and categorize large numbers (>2,000) of erythrocytes which are needed to produce accurate and repeatable reticulocyte counts.

Limitations

• In some systems, light scattering from aggregates of granular platelets produces a pattern that can be miscounted as leukocytes (pseudoleukocytosis).
• Although recent breakthroughs in technology have reduced the cost so that these units are economically justified in most hospitals, they may not be economical in small practices or those that make minimal use of hematology.
• Examination of the blood film is still needed to detect abnormalities such as the presence of left shift, toxicity, reactive lymphocytes, blast cells, mast cells, microfilaria and red cell parasites.

Commercial Laboratory

> Many commercial laboratories offer hematology services at reasonable prices to veterinarians.

> Leading laboratories have state of the art equipment because they are able to amortize the cost over a very large number of samples.

> Counts are performed on automated equipment supplemented by microscopic examination by trained personnel.

Advantages

• Well-run laboratories have quality control programs to ensure accuracy.
• Films are evaluated by personnel who evaluate hundreds of smears daily. They are thus more likely to notice and diagnose rare and unusual abnormalities.
• Identification of bizarre or neoplastic cells is a job for an expert; unusual or highly abnormal samples should be sent to a laboratory with a board-certified veterinary clinical pathologist.
• Red cell parasites and inclusions can also be difficult to differentiate and should be sent for expert confirmation.
• Commercial laboratories can provide additional commentary on unusual findings and consultation with veterinary clinical pathologists.

Limitations

• The main drawback to the use of external laboratories is time; results are not usually available for several hours or until the next day.

• Can be an issue for time-sensitive cases or pre-anesthetic screening.
• Fresh blood smears should be prepared in-clinic and sent with EDTA samples is hematology is performed at reference laboratories.
• Check with your reference laboratory and confirm that blood smears are at least scanned by trained professionals.

• Hematology is species specific. Some laboratories that do primarily human clinical pathology will accept veterinary samples, but this can lead to erroneous results.

• Automated hematology equipment must be re-calibrated for each species or it will produce inaccurate results.
• Likewise, persons evaluating blood smears must be familiar with species differences or they may misclassify white blood cell types and may misinterpret normal variation as a disease condition.
• Large laboratories have many technologists. Each technologist evaluates blood films somewhat differently. The veterinarian cannot assume that all slides are evaluated in the same way.
• Always use a veterinary reference laboratory or one that is familiar with evaluating veterinary samples and has veterinary pathologists on staff.