1a hematology slides


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1a hematology slides

  1. 1. Clinical Pathology • Hematology Leukocytes Erythrocytes Bone Marrow Hemostasis & Platelet abnormalities Cytology • Clinical Chemistry • Integration of hematology, chemistry, cytology
  2. 2. Instructor Mary Anna Thrall Janet Beeler jbeeler@rossvet.edu.kn mthrall@rossvet.edu.kn Office hours: by appointment Dr. Beeler: Pathobiology office #10-08 Dr. Thrall: Pathobiology office no number, on west end, sea side
  3. 3. 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
  4. 4. References • Hematology • Hemostasis • Clinical Chemistry
  5. 5. References • Hematology • Hemostasis • Clinical Chemistry
  6. 6. References
  7. 7. Exams and quizzes Exam 1 - 95 points Exam 2 - 95 points Final and comprehensive - 150 points Take home and in class quizzes – 20 points Lab exam- 40 points Total points possible - 400
  8. 8. 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.
  9. 9. Complete Blood Count Direct measurements • PCV • Hemoglobin • Red cell count • Mean cell volume • White cell count (Total cell count, nucleated cell count)
  10. 10. Complete Blood Count Direct measurements • Plasma protein • Platelet count • Mean platelet volume • Reticulocyte (immature erythrocytes) count • Reticulocyte MCV
  11. 11. Complete Blood Count Microscopic Procedures Differential White cell count (Differential nucleated cell count because includes nucleated erythrocytes) Red cell morphology Platelet morphology, assessment of adequate numbers Reticulocyte count if anemic
  12. 12. Complete Blood Count Calculations Erythrocyte indices Mean corpuscular hemoglobin concentration RDW Absolute nucleated blood cell differential values (% x total nucleated cell count) Absolute reticulocyte count (% reticulocytes x RBC count)
  13. 13. nucleated cell count
  14. 14. 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.
  15. 15. Sample collection & processing
  16. 16. Color coding of stoppers Red - no anticoagulant Blood is expected to clot so that serum can be harvested for biochemical profile and many other tests.
  17. 17. Color coding of stoppers Lavender Contains the anticoagulant EDTA (ethlenediaminetetraacetic acid with a potassium salt) Preserves cell morphology Used to collect blood for CBC
  18. 18. Color coding Green - heparin - specific tests (ie, lead conc) Blue - citrate - coagulation tests Gray - fluoride - inhibits glucose metabolism Red and black - “Sure-sep” tube Used to separate serum from RBCs without transferring serum to different tube
  19. 19. 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.
  20. 20. Tissue contamination results in activation of platelets with resulting clot formation, and erroneously low platelet count.
  21. 21. 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.
  22. 22. 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.
  23. 23. 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.
  24. 24. Blood for biochemical profile Blood allowed to clot for 15 to 30 minutes Centrifuge Separate serum from clot using pipette Refrigerate harvested serum until analyzed Freeze if can’t analyze within two days Some serum enzymes are not stable, but most are.
  25. 25. 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
  26. 26. Hematologic techniques Blood mixing • Important before any analyses - performed because cells settle out. • Manually or with a tilting rack or wheel.
  27. 27. Packed Cell Volume • Percentage of whole blood composed of erythrocytes • Also called “hematocrit” • Measured after centrifugation that maximally packs RBCs
  28. 28. Fill tube 70 - 90% of its length
  29. 29. Buffy coat • Leukocytes • Nucleated erythrocytes • Platelets
  30. 30. Plasma • yellow pigmentation - suggestive of icterus (increased bilirubin concentration in the blood) In large animals, may be due to carotene pigments associated with diet
  31. 31. Plasma • White, opaque - lipemia (chylomicrons) - may be due to postprandial collection or may be due to diseases associated with abnormalities in lipid metabolism
  32. 32. 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.
  33. 33. Plasma protein by refractometry Refractometers 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
  34. 34. 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.
  35. 35. Plasma protein abnormalities Follow up with biochemical measurement of albumin and globulin in serum Decreases may be due to decreased albumin, decreased globulin, or both Increases may be due to increased albumin, increased globulin, or both. Increased albumin always due to dehydration. If total protein and PCV both increased, suggests dehydration If total protein and PCV both decreased, suggests blood loss
  36. 36. 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)
  37. 37. 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.
  38. 38. 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.
  39. 39. 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
  40. 40. BLOOD FILM PREPARATION Stained 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
  41. 41. Blood film preparation • “Push” technique best • Two slides needed. • Increase angle if animal is anemic • Push QUICKLY
  42. 42. 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.
  43. 43. Examination of blood film Success 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.
  44. 44. Nucleated RBC Neutrophil
  45. 45. F E A T H E R E D E D G E
  46. 46. 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
  47. 47. Differential nucleated cell count • Count a minimum of 100 nucleated cells within the counting area • Classify nucleated cells as Segmented neutrophils Band neutrophils Lymphocytes Monocytes Eosinophils Basophils Nucleated RBCs Other
  48. 48. Conversion of % to absolute Multiply 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 µl 80% of cells are segmented neutrophils 80% x 10,000 µl = 8,000 µl segs
  49. 49. Erythrocyte morphology Color Shape Inclusions Arrangement on slide
  50. 50. 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.
  51. 51. 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)
  52. 52. Hematologic techniques performed with automated instrumentation Calculations Hematocrit (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
  53. 53. Mean Cell Hemoglobin Concentration not particularly useful diagnostically Hgb (g/dl) x 100 = MCHC (g/dl) PCV (%) 10 g/dl x 100 = 33.3 g/dl 30% Normal is approx 32 to 36 g/dl in all species except camel family members, which have approx 41 -45 g/dl
  54. 54. Mean Cell Hemoglobin Concentration Increases are always artifactual If 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.
  55. 55. Cell sizing and counting Light 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.
  56. 56. Cell sizing and counting Electronic 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.
  57. 57. 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.
  58. 58. Erythrocyte Size Normal values vary with species Dog: 60 - 72 fl Cat, horse, cow: 39 - 52 fl Sheep: 25-35 fl Llama: 21 - 29 Goat: 15-25 Human: 80 - 100 fl
  59. 59. ERYTHROCYTE SIZE MCV determined electronically Rel # of RBCs Cell volume (fl) MCV
  60. 60. 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.
  61. 61. ERYTHROCYTE SIZE MCV determined electronically Rel # of RBCs MCV RDW
  62. 62. 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.
  63. 63. 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.
  64. 64. Interpretation of reticulocyte count • Dogs: 0 - 60,000/µl • Cats: 0 - 40,000/µl • Cows: 0, but can respond • Horses: Do not release reticulocytes
  65. 65. Reticulocyte interpretation Non regenerative anemia: 0 - 10,000µl Poorly regenerative anemia: 10,000 - 60,000 µl Mild to moderate regeneration: 60,000-200,000 µl Maximal regeneration: > 200,000 µl
  66. 66. 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.
  67. 67. Options for laboratory service • In-house (performed on the premises) • Commercial veterinary laboratory Examples: Idexx, Antech Diagnostics • Human laboratory or community hospital
  68. 68. Advantages of In-house testing • Rapid turnaround time • Control over when tests are performed
  69. 69. 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
  70. 70. 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
  71. 71. 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
  72. 72. Disadvantages of commercial veterinary laboratories Fixed turnaround times Sample transportation logistics Cost of sample transportation Variable quality in pathology support and consultation.
  73. 73. Advantages of human laboratories or hospitals • May be only option in remote areas • Sample transportation may be simple
  74. 74. 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.
  75. 75. 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?
  76. 76. Leukocytes & Leukograms Numeric data + morphologic abnormalities = leukogram Abnormal leukograms can describe pathologic processes (eg, the presence of inflammation) Leukogram + other lab data and clinical findings may lead to diagnosis
  77. 77. Leukocyte Morphology neutrophil monocyte
  78. 78. 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)
  80. 80. Bands vs metas length, width of nucleus
  81. 81. 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.
  82. 82. LYMPHOCYTES Normal with granules Reactive
  83. 83. 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.
  84. 84. monocytes & bands
  85. 85. Monocytes vs metamyelocytes Color of cytoplasm, chromatin pattern
  86. 86. Eosinophils • Functions are related to modulation of immune-complex reactions, modulation of allergic inflammation, defense against parasites, etc. • Prominent pink staining granules in cytoplasm.
  87. 87. Basophils • Basophils contain histamine, heparin, and numerous other proteins. • Increased concentration often related to parasitic infestations.
  88. 88. Lymphocyte Nucleated RBCs Neutrophil Howell Jolly body spherocytes
  89. 89. Nucleated RBC Neutrophil
  90. 90. 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.
  91. 91. 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.
  93. 93. 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.
  94. 94. 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.
  95. 95. 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)
  96. 96. 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.
  97. 97. 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)
  98. 98. 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).
  99. 99. 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.
  100. 100. 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).
  101. 101. 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.