1. DIFFERENTIALS
INTRODUCTION

2. 3a. Given the appropriate specimens, reagent, and equipment, perform four of five blood cell differentials and reticulocyte counts with no more than three instructor assists.
(1) Preparation of Peripheral Blood Smears (2) Staining Techniques (3) Rules for Cell Maturation (4) WBC and Platelet Morphology (5) RBC Morphology (6) Reticulocytes (7) Differential WBC Count (8) Procedure

3. 3a. Given the appropriate specimens, reagent, and equipment, perform four of five blood cell differentials and reticulocyte counts with no more than three instructor assists.

4. Preparation of Peripheral Blood Smear
Used to perform differential white cell count
Specimen Collection
Venipuncture, fingerstick or heelstick
EDTA whole blood less than four hours

5. Making Peripheral Blood Smears
Place slide on gauze
Use Diff-Safe dispenser or hematocrit tube
Place blood 1/3 up from frosted end
Use spreader slide

6. Making Peripheral Blood Smears
Hold the spreader slide at a 30º angle
Slide back toward the opposite end of the slide
When contact is made, the specimen will spread along the spreader slide

7. Push the spreader slide toward the opposite end of the slide

8. Making Peripheral Blood Smears
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9. Criteria Feathered edge NO lines, ridges, or holes
Length of smear - no more then 2/3 the length of the slide
Length too long - decrease specimen, increase angle
Length too short - increase specimen, decrease angle
Speed

10. Staining Techniques Wright’s Stain Methyl alcohol - Fixative
Methylene blue – Nucleus
Eosin - Cytoplasm

11. Staining Techniques Modified Wright’s Stain Glycerin – Freezing RBCs
Giemsa - Parasites

12. Too acidic: Stain Buffers Stains must have a pH of 6.4
RBCs appear bright red
WBC nucleus appears pale blue or colorless

13. Too alkaline: Stain Buffers RBCs appear grey or deep blue
WBCs appear very dark blue- black

14. Staining Methods Stain Jar – excellent for STAT specimens
Automatic – excellent for large volumes

15. Correct Area to Read Diff
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16. Direction of Diff Reading
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17. Rules of Maturation
Size
Cytoplasm
Percentage
Color
Granules
Nucleus - Shape

18. WBC and Platelet Morphology
LEUKOPOIESIS: The production of WBCs
Develop in Bone Marrow
Two groups of WBCs
Granulocytes
Agranulocytes

19. Maturation sequence of granulocytes
Myeloblast
Promyelocyte
Myelocyte
Metamyelocyte (Neutrophilic)
Band Neutrophil (Stab)
Segmented Neutrophil

20. Myeloblast Nucleus = 4/5 of cell 2 or more nucleoli
Cytoplasm = nongranular

21. Promyelocyte Nucleus = round/oval Occupies half or more
Cytoplasm = 1st appearance of distinct granules

22. Myelocyte Last stage capable of cell division
Nucleus = slightly flattened on one side no nucleoli
Cytoplasm =
“Dawn of Neutrophilia”

23. Metamyelocyte
Nucleus =
“kidney Bean Shape”
Cytoplasm

24. Band (Stab) Nucleus = Band or horse-shoe shaped 2 sides run parallel
Cytoplasm = (secondary) Granules stain pink-tan

25. Segmented Neutrophil
Nucleus = 2-5 segments (lobes) connected by thread-like filaments
Cytoplasm = Stains pink-tan (secondary) granules
Highest % of WBC seen
Bacterial Infections

26. Eosinophil Nucleus = Usually 2 lobes
Cytoplasm = Large Orange to bright red. “berry shaped granules
Allergies & Parasitic Infections

27. Basophil Nucleus = 2-4 lobes
Cytoplasm = Specific Purple-Black granules mask the appearance of nucleus.
Allergic Reactions

28. Agranulocytic Series groups: Lymphocytic series Monocytic series
Agranulocytes are placed into three
groups:
Lymphocytic series
Monocytic series
Plasmocytic series
Lymphocytes
Monocyte
Plamocyte

29. Lymphocyte 2nd numerous cell seen Production of Antibodies
Nucleus = round/oval
Cytoplasm = may appear none
Azurophilic Granules
Viral Infections

30. Plasmocyte Nucleus = Eccentrically located
Cytoplasm = small “tapioca-like” globules
Mottled Foamy appearance
B Lymphocytes
Immunoglobulins

31. Monocyte Nucleus = “Brain-like convolutions”
Cytoplasm = Blue-Gray ground glass appearance; vacuoles usually present
Phagocytosis – eats microorganisms

32. Thrombocytes (Platelet)
Important in the coagulation process
Less mature stages rarely seen
Maturation sequence:
Megakaryoblast
Promegakaryocyte
Megakaryocyte
Thrombocyte
Abnormal
Platelets
Platelet
Satellitism

33. Thrombocyte (platelet)
Coagulation Process
Produced directly from the cytoplasm of Megakaryocytes
Round, oval, or spindle shaped
 Platelets

34. Reactive (Atypical) Lymphocyte
Nucleus = Eccentrically located; Nucleoli
Cytoplasm = Sky blue peripheral shading stains deeper near RBCs. May contain vacuoles and appear to flow around RBCs
Infectious Mono and Hepatitis

35. Hypersegmentation Segmented Neutrophil
Nucleus = 6 or more lobes w/ filaments
Vitamin B 12 Deficiency, Folate Deficiency & Pernicious Anemia

36. Pelger-Huet Anomaly Two-lobe connected by a filament (Pince-Nez)
Or One-Lobed (peanutshaped)

37. Toxic Granulation
Occurs in neutrophillic metamyelocytes, bands, and segs
Granules stain blue-black in color
Acute Infections, Drug Poisoning, and Burns

38. Dohle Bodies Cytoplasm = Blue or Grayish “clouds” in neutrophils
Bacterial Infections

39. Auer Rods
Cytoplasm = Cigar or Rod-shaped bodies stain reddish-purple color
Found in Myeloblast and Promyelocyte also in Monocytes

40. Smudge / Basket Cell Formed during blood smear prep
Normal in small numbers

41. Vacuolated Giant Platelet

42. Erythrocytic Maturation Sequence
©2002 MTS, University of Washington Department of Laboratory Medicine
Rubriblast
Prorubricyte
Rubricyte
Metarubricyte
(nRBC)
Diffusely basophilic erythrocyte
(polychromatophilic erythrocyte or
Reticulocyte)
Erythrocyte/Mature RBC

43. RBC Morphological Variations
Anisocytosis: Any variation in size outside the normal RBC diameter.
Microcytosis (microcytes)
Macrocytosis (macrocytes)

44. RBC Morphological Variations
Poikilocytosis: Major deviation from normal shape
Burr cells
Acanthocytes
Crenated RBCs
Tear drop cells
Sickle cells
Spherocytes
Ovalocytes/Elliptocytes
Target cells (codocytes)
Helmet cells (schistocytes)

45. RBC Morphological Variations
Hypochromia
Enlarged central zone of pallor
Polychromatophilia
Larger than mature RBC
Stains pink-blue

46. RBC Morphological Variations
Inclusions:
DNA
Howell-Jolly Bodies
Basophilic Stippling
Cabot Rings
Parasites
Hemoglobin Crystals
Pappenheimer Bodies
RNA
Malarial Gametocytes
Hgb C Crystals

47. Malaria Cabot Ring
Figure-eight like beads of a necklace

48. 3c. Using the procedure, specimens,. reagents, and equipment, perform
3c. Using the procedure, specimens, reagents, and equipment, perform three blood cell differentials with no more than four instructor assists.

49. Examination of blood smear
Quantitative and qualitative analysis of WBCs
Semi-quantitative and qualitative analysis of platelets
Evaluation of the morphological characteristics of RBCs

50. Evaluate RBC Morphology
Examine at least 5 oil immersion fields for size, shape, color, NRBCs and inclusions
Normochromic/normocytic
Hypochromia
Anisocytosis
Poikilocytosis
Immature cells
N/N
Hypochromic
Metarubricyte

51. Reporting Percentage of Cells
Slight: 25%
Moderate: 50%
Marked: > 75%

52. Platelet Estimation 0-6 platelets/oil immersion field
Report as decreased
7-20 platelets/oil immersion field
Report as adequate
>20 platelets/oil immersion field
Report as increased

53. Normal Values Segmented neutrophil: 55-65% Lymphocyte: 20-35%
Basophil: %
Eosinophil: %
Monocyte: %
Band neutrophil: %
Immature: %

54. 3d. Using the procedure, specimens,. reagents, and equipment, perform
3d. Using the procedure, specimens, reagents, and equipment, perform reticulocyte counts with no more than four instructor assists.

55. Principle Non-nucleated immature RBCs retain traces of remnant RNA
Supravital staining
substance appears as chain-like reticulum
# of retics counted per1000 RBCs
expressed as a percentage

56. Calculations
% Reticulocytes = Total # retics per 1000 RBCs 10

57. Sources of Error Evaluating less than 1,000 RBCs
Evaluating more than 1000 RBCs
Confusion with RBC inclusions
Failure to remix after incubation
Incorrect stain:blood ratio
Use of heparinized whole blood

58. 3a. Using the procedure, specimens,. and
3a. Using the procedure, specimens, and equipment, prepare two peripheral blood smears with a feathered edge covering one-half to two-thirds the length of the microscope slide.
(1) Preparation of Peripheral Blood Smears (2) Staining Techniques

59. 3b. Using no reference and visual aids provided,
3b. Using no reference and visual aids provided, perform blood cell differential with a minimum of 70% accuracy.
(1) Rules for Cell Maturation (2) WBC and Platelet Morphology (3) Red Blood Cell Morphology (4) Reticulocytes

60. 3c. Using the procedure, specimens,
3c. Using the procedure, specimens, reagents, and equipment, perform blood cell differentials with no more than four instructor assists.
(1) Differential White Blood Cell Count (2) Normal Values

61. 3d. Using the procedure, specimens,
3d. Using the procedure, specimens, reagents, and equipment, perform reticulocyte counts with no more than four instructor assists.
(1) Clinical Significance (2) Principle (3) Equipment, Reagents, and Supplies (4) Procedure (5) Calculations (6) Normal Values (7) Sources of Error (8) ORM Group Discussion

62. ©2002 MTS, University of Washington Department of Laboratory Medicine
Hypochromasia
©2002 MTS, University of Washington Department of Laboratory Medicine

63. ©2002 MTS, University of Washington Department of Laboratory Medicine
Spherocytosis
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64. ©2002 MTS, University of Washington Department of Laboratory Medicine
Spherocytosis
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65. ©2002 MTS, University of Washington Department of Laboratory Medicine
Teardrop Cells
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66. ©2002 MTS, University of Washington Department of Laboratory Medicine
Target Cells
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67. ©2002 MTS, University of Washington Department of Laboratory Medicine
Stomatocytes
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68. ©2002 MTS, University of Washington Department of Laboratory Medicine
Sickle Cells
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69. ©2002 MTS, University of Washington Department of Laboratory Medicine
Basophilic Stippling
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70. ©2002 MTS, University of Washington Department of Laboratory Medicine
Reactive Lymphocyte
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71. ©2002 MTS, University of Washington Department of Laboratory Medicine
Reactive Lymphocyte
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72. ©2002 MTS, University of Washington Department of Laboratory Medicine
Poikilocytosis
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73. Poikilocytosis/Schistocytes
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74. ©2002 MTS, University of Washington Department of Laboratory Medicine
Abnormal Platelets
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75. ©2002 MTS, University of Washington Department of Laboratory Medicine
Platelet Satellitism
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76. ©2002 MTS, University of Washington Department of Laboratory Medicine
Plasmocyte
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77. ©2002 MTS, University of Washington Department of Laboratory Medicine
Metarubricyte
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78. Normochromic/Normocytic
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79. ©2002 MTS, University of Washington Department of Laboratory Medicine
Monocyte
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80. ©2002 MTS, University of Washington Department of Laboratory Medicine
Lymphocytes
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81. ©2002 MTS, University of Washington Department of Laboratory Medicine
Hypochromasia
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82. ©2002 MTS, University of Washington Department of Laboratory Medicine
Hypersegmentation
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83. ©2002 MTS, University of Washington Department of Laboratory Medicine
Hypersegmentation
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84. ©2002 MTS, University of Washington Department of Laboratory Medicine
Ovalocyte
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85. ©2002 MTS, University of Washington Department of Laboratory Medicine
Macrocytes
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86. ©2002 MTS, University of Washington Department of Laboratory Medicine
Burr Cells
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87. ©2002 MTS, University of Washington Department of Laboratory Medicine
Howell-Jolly Bodies
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88. Target Cells/Metarubricyte
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89. Polychromatophilic/Diffusely Basophilic RBC
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90. ©2002 MTS, University of Washington Department of Laboratory Medicine
Malarial Gametocytes
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91. ©2002 MTS, University of Washington Department of Laboratory Medicine
Hgb C Crystals
©2002 MTS, University of Washington Department of Laboratory Medicine

92. ©2002 MTS, University of Washington Department of Laboratory Medicine
Retic and Heinz Bodies
This project was funded at $3,000,000 (100% of its total cost) from a grant awarded under the Trade Adjustment Assistance Community College and Career Training Grants, as implemented by the U.S. Department of Labor’s Employment and Training Administration. Rogue Community College is an equal opportunity employer/program. Auxiliary aids and services, alternate form and language services are available to individuals with disabilities and limited English proficiency free of cost upon request.
This work is licensed under a Creative Commons Attribution 4.0 International License.
©2002 MTS, University of Washington Department of Laboratory Medicine