Lecture 2 - Hematology Instruments and Blood Cell Measurements

what is a Complete Blood Count (CBC)?

• hematocrit, hemoglobin, RBC count

• Erythrocyte indices - MCV, MCH, MCHC, and sometimes RDW

what is a Leukocyte count?

• total leukocyte count

• differential leukocyte count

what does a CBC involve?

• Erythrocyte parameters

• Leukocyte counts

• Platelet counts

what are the benefits of in-house Analyzers?

• rapid info: emergencies, client service

• analysis of fresh samples: rebleed if needed, avoids sample deterioration

• info available after-hours and on weekends

• cost effective with sufficient numbers of samples

what are the two different types of Technology?

• Electrical Impedance Method

• Laser Flow Cytometry Method

What is the electrical Impedance method?

measures how a material or object resists the flow of electrical current

what is change in resistance proportional to in terms of the electrical impedance method?

to cell volume

What is the Coulter Impedance Method?

Change in resistance is proportional to cell volume

What do platelet and RBC volume histograms provide?

a visual representation of the size distribution of RBCs and platelets, offering insights into cell abnormalities and potential diseases that mere cell counts might miss

what do impedance instruments do?

count and size cells, provide cell volume distributions

what specifically do impedance instruments measure?

hemoglobin (Hb) spectrophotometrically

what specific values are reported with impedance instruments?

• Hematocrit (HCT)

• Red Blood Cell (RBC)

• Hemoglobin (Hb)

• Mean Corpuscular Volume (MCV)

• Mean Corpuscular Hemoglobin (MCH)

• Mean Corpuscular Hemoglobin Concentration (MCHC)

• Red Cell Distribution Width (RDW)

what is hematocrit?

blood test that measures the percentage of red blood cells in your blood

what is hemoglobin?

protein found in red blood cells, responsible for carrying oxygen from the lungs to the body’s tissues and organs, and also transporting carbon dioxide back to the lungs

what is Mean Corpuscular volume (PCV)?

a blood test that measures the average size of your red blood cells

what is the mean corpuscular hemoglobin (MCH)?

measure of the average amount of hemoglobin in a single red blood cell

what is the Mean Corpuscular Hemoglobin Concentration (MCHC)?

a measure of the average concentration of hemoglobin within a red blood cell

what is the Red cell Distribution Width (RDW)?

blood test that measures the variation in size and volume of RBCs (erythrocytes)

what can impedance instruments NOT do?

• cannot determine intracellular characteristics of cells

• cannot do platelet counts accurately in cats

• Cannot perform reticulocyte counts

what are some general things with impedance instruments?

• total WBC (all species) and generally platelet counts are generally reported

• Excellent precision

• generally, less expensive than laser flow instruments

what kind of differential do impedance instruments have?

only a 3-part differential based on cell size alone (limited accuracy)

what is laser flow cytometry?

technique that uses lasers to analyze the physical and chemical characteristics of individual cells or particles suspended in a fluid

how does laser flow cytometry work?

involves passing a stream of cells through one or more laser beams, measuring how the light scatters and how the cells fluoresce, and then using this data to characterize the cell population

what does forward scatter of laser flow cytometry indicate?

cell size

what does side scatter of laser flow cytometry indicate?

complexity/granularity

what do detectors measure with laser flow cytometry?

placed in front of the laser beam to measure forward scatter, and at angles (typically 90 degree) to the laser beam to detect light scattered to the side

what does the forward scatter detector measure?

the cell’s shadow and is a relative indicator of the cell’s size

what does the side scatter detector measure?

• also called right angle or orthogonal scatter

• a measure of the cell’s internal complexity or granularity

what cells usually have side scatter?

• neutrophils: multi-lobed nucleus

• Granules: high amounts of side scatter

what cells have lower side scatter?

Lymphocytes with their high nucleus:cytoplasm ratio and scant cytoplasm

how does fluorescence help with laser flow cytometry?

may help be used to ID cell types

what does Laser Flow Cytometry determine?

• number, size, and internal complexity of cells

• counts and classifies cells based on extinction of light and scattered light

what values do Laser Flow Cytometry provide?

HCT, RBC, Hb, MCV, MCH, MCHC, RDW

what kind of differential does laser flow cytometry have?

• total WBC

• 5 part differential counts, although basophils are not accurately counted in dogs

What other important counts does Laser Flow Cytometry provide?

• Reticulocyte count

• platelet counts, mean platelet value (MPV), Platelet Distribution Width (PDW), and Procalcitonin (PCT)

what is mean platelet value (MPV)?

measurement of average size of platelets in a blood sample

what is the platelet distribution width (PDW)?

assesses the variability in the size of your blood platelets, which are crucial for blood clotting

what is Procalcitonin (PCT)?

protein produced by various cells in response to bacterial infection; test measures the level of this in the blood

RBC count characteristics?

• little value by itself

• used to calculate Hct electronically

• used to calculate MCV and MCH manually

PCV and Hct determination?

• Centrifugation - packed cell volume (PCV)

• automated cell counters calculate the hematocrit (Hct) from the MCV and RBC count

what is the equation to determine the Hct %?

MCV x RBC / 10

how do you determine Hemoglobin?

Spectrophotometric assay

what reagent is used for spectrophotometric assay to determine hemoglobin?

Sodium lauryl sulfate (SLS)

what does the SLS reagent do?

• lyses erythrocytes

• oxidizes Hb to MetHb and forms a stable SLS - MetHb product

what things can falsely increase hemoglobin

• lipemia

• Heinz bodies

• Nuclei in non-mammals

what is spectrophotometry measuring?

light absorbance; more light absorbed through = higher absorbance value (correlated with protein conc.)

what are the Erythrocyte indices?

• MCV: mean cell volume

• MCH: mean cell hemoglobin

• MCHC: mean corpuscular hemoglobin conc.

• RDW: Red cell distribution width

what are the units of MCV?

femtoliters

how many femtoliters is the average volume of a single erythrocyte?

10^-15 liter

how is MCV determined?

directly in hematology analyzers

what units represent Mean cell hemoglobin?

picogram = 10^-12 gram

when are low values of mean cell hemoglobin reported in?

severe iron deficiency anemia (low MCV and low internal hemoglobin concentration)

how do you determine MCHC?

by calculation; reported as g/dL of packed erythrocytes (NOT g/dL whole blood)

what is the calculation for MCHC?

Hb/HCT x 100

what is the equation for RDW?

SD of erythrocytes volumes / MCV X 100

anisocytosis?

variation of size cell

what is spuriously increased RDW?

• erythrocyte agglutination

• platelets counted in erythrocyte histogram of severely anemic patients

what is important about reticulocyte counts in cats?

• verify which reticulocytes are counted in cats

• Only aggregate reticulocytes are counted in most instruments

what are aggregate reticulocytes?

• immature RBC

• less mature, containing large amounts of RNA that appear as blue clumps or strands when stained with a vital stain like new methylene blue

what are punctate reticulocytes?

• slightly more mature than aggregate retics

• containing only small amounts of RNA that appear as dots

what type of reticulocytes are found in cats?

• aggregate retics

• Punctate retics

what happens with spurious reticulocytosis?

erythrocyte parasites containing RNA and DNA, e.g., hemotropic mycoplasmas, Babesia species

what typically is involved with spurious reticulocytosis?

• Howell-Jolly bodies (micronuclei)

• Nucleated erythrocytes

• Large immature platelets or platelet clumps

• Heinz bodies with nonspecific fluorescence

• Autofluorescence (drugs, porphyrin)

• Leukocytosis - uncommon

what creates false platelet counts?

• platelet aggregates can result in false thrombocytopenia

• platelet activation during blood collection and handling (especially in cats)

• Verify platelet counts, especially thrombocytopenia by stained blood film examination

• automated platelet counts often unreliable in cats, especially using impedance counters

what is the mean platelet volume (MPV) reference interval for cats?

11.0-18.1

what is the mean platelet volume (MPV) reference range for dogs?

6.7 - 11.1

what is the mean platelet volume (MPV) reference range for horses?

5.9 - 9.9

what is the mean platelet volume (MPV) reference range for cattle?

4.5 - 7.5

what causes spuriously increased MPV?

• platelet aggregates

• storage of blood at 5 C

what happens if nucleated erythrocytes in blood?

• counted as leukocytes by manual and most automated cell counters

• calculate corrected total leukocyte count before calculating absolute differential leukocyte counts

what is the corrected white blood cell count calculation?

Machine WBC x 100/100 + NRBC = Corrected WBC

what does old blood samples cause with the values?

• Erythrocyte swelling (MCV incr., HCT incr., MCHC decreased)

• Platelet aggregation (MPV incr. and platelet count decreased)

• Cell lysis of all cell types - counts decreased

what happens if platelet, leukocyte, erythrocytes aggregates?

• number of cells/microliter of blood are decreased

• erythrocyte aggregates MCV increase, electronic HCT decrease, MCHC increase

• platelet aggregrates may be counted as leukocytes

• MPV is increased with platelet aggregates

• collection in citrate may reduce platelets and leukocytes aggregates

what happens to blood values with clot formation?

all cell types decrease

what do cryoglobulins do to bloodwork?

may be counted as leukocytes or platelets

what do cryoglobulins do?

precipitate below body temp

when there is hemolysis in a blood sample what happens?

• MCHC increased

• in vivo - intravascular hemolysis

• in vitro - sample handling and lipemia

when there is lipemia what happens with the blood sample cell measurements?

• Hb and MCHC increased

• may increase leukocyte or platelet counts

when Heinz bodies are present in sample what happens to the cell measurements?

• Hb and MCHC increased

• sometimes total leukocyte counts are increased

• reticulocyte counts may be increased