Clinical Pathology• Hematology

LeukocytesErythrocytesBone MarrowHemostasis & Platelet abnormalities

Cytology

• Clinical Chemistry

• Integration of hematology, chemistry, cytology

InstructorMary Anna Thrall

Janet Beelerjbeeler@rossvet.edu.knmthrall@rossvet.edu.kn

Office hours:by appointment

Dr. Beeler: Pathobiology office #10-08Dr. Thrall: Pathobiology office no number,

on west end, sea side

Course Objectives• Understand underlying pathophysiology• Understand how tests are selected• Interpret and integrate test results

• Understand Technology to generate data

Use laboratory data to MAKE A DIAGNOSIS

References

• Hematology• Hemostasis • Clinical Chemistry

References• Hematology• Hemostasis • Clinical Chemistry

References

Exams and quizzesExam 1 - 95 pointsExam 2 - 95 pointsFinal and comprehensive - 150 points

Take home and in class quizzes – 20 points

Lab exam- 40 points

Total points possible - 400

Learning objectives – Section 1• Describe components of a CBC and

recognize the information provided by each.

• Be able to calculate absolute values of various types of leukocytes if the differential leukocyte count (percentage), and the total nucleated cell count is provided.

• Be able to calculate the absolute reticulocyte count based on % of RBCS that are retics, and RBC count.

Complete Blood CountDirect measurements

• PCV• Hemoglobin• Red cell count• Mean cell volume

• White cell count (Total cell count, nucleated cell count)

Complete Blood CountDirect measurements

• Plasma protein

• Platelet count• Mean platelet volume

• Reticulocyte (immature erythrocytes) count• Reticulocyte MCV

Complete Blood CountMicroscopic Procedures

Differential White cell count (Differential nucleated cell count because includes nucleated erythrocytes)

Red cell morphologyPlatelet morphology, assessment of

adequate numbersReticulocyte count if anemic

Complete Blood CountCalculations

Erythrocyte indicesMean corpuscular hemoglobin concentrationRDW

Absolute nucleated blood cell differential values(% x total nucleated cell count)

Absolute reticulocyte count (% reticulocytes x RBC count)

Learning objectives, Section 2• Remember the types of collection tubes

that are used for CBC, biochemical profile, coagulation profile, and glucose when serum can’t be separated from clot.

• Recognize errors that can be introduced when incorrect tubes, collection methods, and storage methods are used.

Sample collection & processing

Color coding of stoppersRed - no anticoagulant

Blood is expected to clot so that serum can be harvested for biochemical profile and many other tests.

Color coding of stoppersLavenderContains the anticoagulant EDTA(ethlenediaminetetraacetic acid with a

potassium salt)Preserves cell morphologyUsed to collect blood for CBC

Color codingGreen - heparin - specific tests (ie, lead conc)Blue - citrate - coagulation testsGray - fluoride - inhibits glucose metabolismRed and black - “Sure-sep” tube

Used to separate serum from RBCs without transferring serum to different tube

Technique for filling tubes• “Clean” venipuncture with no tissue

contamination from large vein, such as jugular or cephalic in large dogs.

• CBC and biochemical profile requires approximately 5 ml blood. Pediatric (small) tubes are available.

Tissue contamination results in activation of platelets with resulting clot formation, and erroneously low platelet count.

Techniques for filling tubes• Don’t force blood into tube - don’t use

anything smaller than a 20 G needle for filling.

• Tubes are vacuum tubes - can utilize the vacuum or can actually take the stopper off and fill.

• If using syringe to collect blood, then filling tubes, work quickly, filling tube(s) that contain anticoagulant first.

Ratio of blood to anticoagulant is designed to be appropriate by

amount of vacuum.

• Inadequate filling of purple top tube results in excess EDTA, which causes erythrocytes to shrink, resulting in an erroneous decrease in PCV and MCV.

Sample handling procedures• Blood for CBC - either analyze within one

hour, or make blood film and refrigerate tube (Don’t refrigerate blood film -condensation causes cells to lyse)

• Don’t allow to freeze - freezing lyses cells• If blood sets at room temperature for 24

hours or more, erythrocytes swell, resulting in increase in MCV.

Blood for biochemical profileBlood allowed to clot for 15 to 30 minutes CentrifugeSeparate serum from clot using pipetteRefrigerate harvested serum until analyzedFreeze if can’t analyze within two daysSome serum enzymes are not stable, but

most are.

Basic Hematologic Techniques• Blood mixing• Packed cell volume by centrifugation• Plasma protein estimation by refractometry• Leukocyte concentration by Unopette• Preparation of blood film• Differential leukocyte count • Blood film examination

Hematologic techniques

Blood mixing• Important before any analyses -

performed because cells settle out.• Manually or with a tilting rack or wheel.

Packed Cell Volume• Percentage of whole blood composed of

erythrocytes• Also called “hematocrit”• Measured after centrifugation that

maximally packs RBCs

Fill tube 70 - 90% of its length

Buffy coat• Leukocytes• Nucleated erythrocytes• Platelets

Plasma• yellow pigmentation - suggestive of

icterus (increased bilirubin concentration in the blood) In large animals, may be due to carotene pigments associated with diet

Plasma• White, opaque - lipemia (chylomicrons) -

may be due to postprandial collection or may be due to diseases associated with abnormalities in lipid metabolism

Plasma• Red coloration - due to presence of

hemoglobin in plasma• Result of hemolysis• May be in-vitro due to technique or

presence of lipemia• May be in-vivo due to hemolytic anemia

(intravascular hemolysis)• IF PCV not decreased, likely in-vitro.

Plasma protein by refractometryRefractometers estimate the concentration

of solute in fluid, since solute bends slight passing through the fluid proportionate to the solute concentration.

Measures the refractive index relative to distilled water.

Used in clinical diagnostics to estimate plasma protein estimation & urine specific gravity

Plasma protein by refractometry• Protein concentration is an estimate,

assuming that other solutes in the serum are present in normal concentrations.

• Lipemia will artificially increase the estimate.

• Urea, glucose, cholesterol may also influence (artificially increase) the estimate.

Plasma protein abnormalitiesFollow up with biochemical measurement of albumin

and globulin in serumDecreases may be due to decreased albumin,

decreased globulin, or bothIncreases may be due to increased albumin,

increased globulin, or both. Increased albumin always due to dehydration.If total protein and PCV both increased, suggests

dehydrationIf total protein and PCV both decreased, suggests

blood loss

Total Leukocyte concentration• Actually Total nucleated cell count• Technique detects all nuclei in solutions

from which RBCs have been removed by lysis (or in some systems, centrifugation)

Unopette system• Also need hemocytometer counting

chamber and microscope.• Unopette capillary is used to transfer 20µl

blood into 1.98 ml acetic acid diluent, resulting in a 1:100 dilution.

• Acid lysis cytoplasmic membranes, eliminating RBCs, leaving behind only nuclei.

• Incubate, mix, place onto hemocytometer.

Unopette system• After filling hemocytometer system, allow

to settle, then examine, using the 10x objective of the microscope. The volume of fluid present is 0.9 µl. Nuclei are counted in all 9 grids. After counting nuclei, add 10% to yield the number of cells in 1.0 µl. Multiply the result by 100 (the dilution factor). This results in the total number of nuclei/µl.

Total leukocyte (nucleated cell) count

By itself is NOT very useful for interpretative purposes. It is used

primarily to CALCULATE the concentration of specific leukocyte types, which is determined after performing the

DIFFERENTIAL CELL COUNT

BLOOD FILM PREPARATIONStained blood film is essential tool for

1) determining concentration of individual leukocyte types (differential cell count)

2) evaluating morphologic abnormalities of leukocytes, erythrocytes, and platelets

Blood film preparation• “Push” technique best• Two slides needed. • Increase angle if animal is anemic• Push QUICKLY

Staining the slide• Wright Stain• Wright-Giemsa Stain

• Variety of quick stains that are modifications of Wright stain are available, such as “Diff-Quick”

• Best to use “dipping” procedures.

Examination of blood filmSuccess depends on:

• Quality of blood film preparation• Examination of correct part of blood film• Recognition of morphologic abnormalities• Recognition of artifacts• Ability to interpret diagnostic significance

of morphologic abnormalities. For example, spherocytes = IMHA.

Nucleated RBC

Neutrophil

FEATHERED

EDGE

Examine with 50x or 100 x objective

• Systematic examination of all three major cell types (RBCs, leukocytes, platelets)

• Differential count of leukocytes• Evaluation of RBC morphology• Estimation of platelet concentration & size

Differential nucleated cell count• Count a minimum of 100 nucleated cells within

the counting area• Classify nucleated cells as

Segmented neutrophilsBand neutrophils

LymphocytesMonocytesEosinophilsBasophils

Nucleated RBCsOther

Conversion of % to absoluteMultiply the total nucleated cell

concentration by the percentage of each leukocyte type to yield the absolute

concentration of each type of nucleated cell within the blood sample.

Eg, total nucleated cell count = 10,000 µl80% of cells are segmented neutrophils

80% x 10,000 µl = 8,000 µl segs

Erythrocyte morphologyColorShapeInclusionsArrangement on slide

Platelet evaluation• Estimate concentration• 6 - 10 platelets per oil immersion field,

depending on microscope’s field of view• Remember to examine feathered edge for

clumps if platelets appear decreased.• When platelets approach the size of RBCs

they are termed macroplatelets or giant platelets.

Hematologic techniques performed with automated instrumentation

Cell particle counting and sizing• RBC count (x106/µl• MCV (fl)• Nucleated cell count (µl)• Differential cell count (µl)• Platelet count (µl)• Reticulocyte count and size (µl, fl)

Spectrophotometry• Hemoglobin concentration (g/dl) (usually approx

1/3 of the PCV)

Hematologic techniques performed with automated instrumentation

CalculationsHematocrit (PCV)

MCV x RBC = HCT (PCV)10

MCV 70 FL x 7.0 RBC = HCT 49%10

Also, MCV can be calculated if PCV and RBC are known. PCV/RBC x 10 = MCV

Mean Cell Hemoglobin Concentration

not particularly useful diagnosticallyHgb (g/dl) x 100 = MCHC (g/dl)

PCV (%)

10 g/dl x 100 = 33.3 g/dl30%

Normal is approx 32 to 36 g/dl in all species except camel family members, which have approx 41 -45 g/dl

Mean Cell Hemoglobin ConcentrationIncreases are always artifactualIf increased, is due to erroneously high hemoglobin

reading due to hemolysis, lipemia, or presence of Heinz bodies (pieces of denatured hemoglobin as a result of oxidation).

Decreases may be due to iron deficiency, but not unless very severe.

Usually decreases due to presence of many reticulocytes that are still making hemoglobin, usually associated with a regenerative anemia.

Cell sizing and countingLight scatter measurement (Flow)

Cells are passed through a flow cell that is intersected by a focused laser beam.

Physical properties of the cells scatter light to different degrees and different angles relative to the light source. Scatter events are counted to derive the cell concentration. The degree of scatter in the direction of the light beam, known as forward angle scatter, is proportional to the size of the cell. Light scattered in other angles is correlated with other cellular properties, leading to the ability to differentiate cell types.

Cell sizing and countingElectronic cell counting (impedence)

Cells are suspended in an electrolyte medium that conducts electricity.

Cells are relatively poor conductors of electricity.

Deflections in current are proportional to the size of the cell. Cells are thus identified and measured.

Size distribution curves• Established for each population of cells

(eg, erythrocytes, platelets).• Must be set for each species, since

erythrocyte sizes are so variable for different species of domestic animals.

• Primary reason that instruments set up to measure human blood are inappropriate for domestic animals.

Erythrocyte SizeNormal values vary with species

Dog: 60 - 72 flCat, horse, cow: 39 - 52 flSheep: 25-35 flLlama: 21 - 29Goat: 15-25

Human: 80 - 100 fl

ERYTHROCYTE SIZE

MCV determined electronically

Rel # ofRBCs

Cell volume (fl)

MCV

Red cell distribution width (RDW)• Describes the relative width describes

the relative width of the size distribution curve.

• It is the standard deviation of most of the erythrocytes divided by the MCV. The tails of the erythrocyte distribution are usually excluded from this calculation.

ERYTHROCYTE SIZE

MCV determined electronically

Rel # ofRBCs

MCV

RDW

Reticulocyte concentration• Can now be determined by Flow technology• Can be determined manually• Immature erythrocytes (reticulocytes) still have

organelles for protein synthesis and aerobic metabolism (ribosomes and mitochondria). Certain stains cause these residual organelles to aggregate, resulting in clumped material that can be seen.

• Reticulocytes are polychromatic RBCs when stained with Wrights stain.

Reticulocyte count• New Methylene Blue• Brilliant cresyl blue• 1000 RBCs are counted. The percentage

of RBCs that are reticulocytes • Percentage is multiplied by the RBC count

to obtain an absolute reticulocyte concentration.

Interpretation of reticulocyte count

• Dogs: 0 - 60,000/µl• Cats: 0 - 40,000/µl• Cows: 0, but can respond• Horses: Do not release reticulocytes

Reticulocyte interpretationNon regenerative anemia: 0 - 10,000µlPoorly regenerative anemia: 10,000 - 60,000 µlMild to moderate regeneration: 60,000-200,000 µlMaximal regeneration: > 200,000 µl

Reticulocyte maturation• Dogs - 24 -48 hours from release to

maturation• Cats - Aggregate reticulocytes become

punctate reticulocytes. Punctate forms are not polychromatophilic with Wrights stain, and are not counted in the reticulocyte count. Aggregates become punctates in approx 12 hours. Punctates persist for 12 days.

Options for laboratory service• In-house (performed on the premises)• Commercial veterinary laboratory

Examples: Idexx, Antech Diagnostics• Human laboratory or community hospital

Advantages of In-house testing• Rapid turnaround time• Control over when tests are performed

Disadvantages of In-House tests• Cost of equipment, technician• Expertise required for quality assurance,

trouble-shooting • Will still need to send out some samples

for specialized tests • Will need to consult with clinical

pathologist on difficult blood films, bone marrow aspirates, and cytology samples

In-House Testing considerations• $25,000 to set up hematology and chemistry

systems. Consider instrument amortization, supplies, personnel, training, cost of quality assurance program, time for supervision of tech)

• Practice volume adequate to make it profitable?• Biochemical profiles usually more expensive ($3

per test vs $25 for complete profile)• Hiring and retaining technician that is able,

willing, interested, & will seek consultation• Commitment to quality assurance

– Monitoring of equipment accuracy and precision

Advantages of commercial veterinary laboratories

• May be less expensive• Complete menu of testing services• Professional support of technical

performance, quality control, species differences

• Clinical pathologist usually on-site to review abnormalities, and for consultations

Disadvantages of commercial veterinary laboratories

Fixed turnaround timesSample transportation logisticsCost of sample transportationVariable quality in pathology support and

consultation.

Advantages of human laboratories or hospitals

• May be only option in remote areas• Sample transportation may be simple

Disadvantages of human laboratories or hospitals

• Instrumentation not modified for animals (eg, difference in RBC size will result in significant abnormalities in hemogram)

• Technicians and pathologist not familiar with normal and abnormal blood cells

• Turn around time may be increased due to low priority of animal samples.

Out-House testing considerations

• Community hospital not usually viable option. Species specific testing is critical.

• Sample pick up serviceconvenient?• Appropriate turn-around time? - most labs are

located in large cities on either coast, and Chicago. Results usually faxed or e -mailed.

• Telephone consultation available?

Leukocytes & LeukogramsNumeric data + morphologic abnormalities =

leukogramAbnormal leukograms can describe pathologic

processes (eg, the presence of inflammation)Leukogram + other lab data and clinical

findings may lead to diagnosis

Leukocyte Morphology

neutrophil

monocyte

Common blood leukocytes• Neutrophils - participate in inflammatory

responses by migrating into tissue sites and phagocytizing and killing organisms.

• Immature neutrophils released from bone marrow when inflammation present

(Band neutrophils, metamyelocytes)

NEUTROPHILS&

BANDS

• CONSTRICTION, LENGTH OF NUCLEUS?

Bands vs metaslength, width of nucleus

Lymphocytes• Numerous lymphocyte subpopulations, such

as B- cells (responsible for humoral immunity), T- cells (responsible for cell-mediated immunity), large granular lymphocytes (null cells or T cells), etc.

LYMPHOCYTES

Normal with granules Reactive

Monocytes• Monocytes migrate into tissues to become

macrophages. Functions are many, including phagocytosis, presentation of antigens to T-lymphocytes, iron recycling, cytokine production, etc.

• May be confused with band neutrophils on blood films.

monocytes & bands

Monocytes vs metamyelocytesColor of cytoplasm, chromatin pattern

Eosinophils• Functions are related to modulation of

immune-complex reactions, modulation of allergic inflammation, defense against parasites, etc.

• Prominent pink staining granules in cytoplasm.

Basophils• Basophils contain histamine, heparin, and

numerous other proteins. • Increased concentration often related to

parasitic infestations.

Lymphocyte

Nucleated RBCs

Neutrophil

Howell Jolly body

spherocytes

Nucleated RBC

Neutrophil

Data Interpretation (Chapter 3)Reference ranges (intervals) (normal values)To recognize abnormal results, normal results

must be established, using a population of apparently healthy animals.

Species, age, breed, gender, pregnancy status, geographical location, etc.

The more animals that are sampled, the more accurate the reference interval.

Reference intervals• In some situations, extreme values on

either end should be discarded to increase the sensitivity of the test. These are called “outliers”.

• Simple rule: If the difference between the highest value and the second highest value in this population exceeds one-third of the total range, eliminate it.

FREQUENCY

BLOOD GLUCOSE CONCENTRATION (MG/DL)

Reference intervals• If the data are normally distributed (Bell-

shaped curve), mean and standard deviation are calculated, and the central 95% of values (mean + 2 SD) is considered the reference interval.

• If less than 40 animals are used, then reference interval is the observed range of values that remains after outliers.

Reference Intervals• A few (5%) healthy animals will have results

SLIGHTLY outside of the reference interval. • If result is FAR outside the reference interval,

it probably indicates an abnormality. • Slight abnormalities in some test results, such

as electrolytes, are more significant than slight abnormalities in others, such as serum enzyme activities.

Data interpretation• Use signalment, history, physical exam, and

laboratory data.• Results may exclude some differential diagnoses,

may suggest additional differential diagnoses, or may be diagnostic for a specific disease.

• Results that are most outside of the reference interval are likely the most significant (possibly w/ exception of enzymes)

Data interpretation• Pattern Recognition of abnormal results is

critical for making diagnoses.• For example, an increased BUN and a urine

specific gravity indicating that animal is not concentrating urine is very suggestive of renal disease. An increased BUN with a concentrated urine, indicates that animal is dehydrated or had blood loss into GI tract or other very high protein meal.

Sensitivity of a test• Measure of the frequency with which the

test result will be abnormal in animals with the disease for which the test is being used. If the sensitivity of PCR for lymphoma is 91%, then 91% of animals with lymphoma will have a positive result. (and 9% will have a false negative result)

Specificity of a test• Measure of the frequency with which a

test result will be normal in animals without the disease one wishes to detect.

• If the specificity of a test is 95%, then 95% of unaffected animals will have normal results. (and 5% will have false positive results).

Sensitivity and Specificity• Established by applying the test to animals

with known disease status. IE, known to have or not have the disease in question.

Predictive value of a test• Reliability of a test to detect whether or

not an animal has a certain disease.

• Determined by using both the sensitivity and specificity of a test. (See pages 49 -51).

Quality control• Accuracy - gauge of how close the result is to

the true value for that test.• Precision - gauge of how repeatable the result is

when assaying the same sample.• Quality control programs assure both accuracy

and precision. This is done by assaying control samples on which the correct result is known -control samples are commercially available.

• Problems may be due to instrument, reagents or operator error.

• CRITICAL that your laboratory has a quality control program.