DIFFERENTIALS INTRODUCTION

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

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.

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

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

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

Push the spreader slide toward the opposite end of the slide

Making Peripheral Blood Smears http://www.youtube.com/watch?v=JMd2 RScYu0M&feature=related

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

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

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

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

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

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

Correct Area to Read Diff ©2002 MTS, University of Washington Department of Laboratory Medicine

Direction of Diff Reading ©2002 MTS, University of Washington Department of Laboratory Medicine

Rules of Maturation Size Cytoplasm Percentage Color Granules Nucleus - Shape

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

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

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

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

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

Metamyelocyte Nucleus = “kidney Bean Shape” Cytoplasm

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Vacuolated Giant Platelet

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

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

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)

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

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

Malaria Cabot Ring Figure-eight like beads of a necklace

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.

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

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

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

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

Normal Values Segmented neutrophil: 55-65% Lymphocyte: 20-35% Basophil: 0-1% Eosinophil: 2-4% Monocyte: 2-6% Band neutrophil: 3-7% Immature: 0-1%

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.

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

Calculations % Reticulocytes = Total # retics per 1000 RBCs 10

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

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

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

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

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

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

©2002 MTS, University of Washington Department of Laboratory Medicine Spherocytosis ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Spherocytosis ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Teardrop Cells ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Target Cells ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Stomatocytes ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Sickle Cells ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Basophilic Stippling ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Reactive Lymphocyte ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Reactive Lymphocyte ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Poikilocytosis ©2002 MTS, University of Washington Department of Laboratory Medicine

Poikilocytosis/Schistocytes ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Abnormal Platelets ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Platelet Satellitism ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Plasmocyte ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Metarubricyte ©2002 MTS, University of Washington Department of Laboratory Medicine

Normochromic/Normocytic ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Monocyte ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Lymphocytes ©2002 MTS, University of Washington Department of Laboratory Medicine

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

©2002 MTS, University of Washington Department of Laboratory Medicine Hypersegmentation ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Hypersegmentation ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Ovalocyte ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Macrocytes ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Burr Cells ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Howell-Jolly Bodies ©2002 MTS, University of Washington Department of Laboratory Medicine

Target Cells/Metarubricyte ©2002 MTS, University of Washington Department of Laboratory Medicine

Polychromatophilic/Diffusely Basophilic RBC ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Malarial Gametocytes ©2002 MTS, University of Washington Department of Laboratory Medicine

©2002 MTS, University of Washington Department of Laboratory Medicine Hgb C Crystals ©2002 MTS, University of Washington Department of Laboratory Medicine

©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