Special Stains in Haematology

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Hematology Review : 

Hematology Review By Felicia Magee Tardy, M.S., MT (ASCP) Department of Clinical Pathology University of Mississippi Medical Center Jackson, Mississippi

Introduction : 

Introduction Hematology: the study of blood and blood forming tissues. Blood consists of 55% plasma and 45% formed elements. Formed elements include erythrocytes, leukocytes, and thrombocytes.

Erythrocytes : 

Erythrocytes Normal range 4.2-5.5 million per mm3 in adults. Biconcave shape. Diameter 7 microns. Cells for transport of O2 and CO2. Life span 120 days.

Leukocytes : 

Leukocytes Normal range 4 - 11 thousand per mm3 in adults. Five types. Size 8-20 microns. Involved in fighting infection, combatting allergic reactions, and immune responses.

Thrombocytes : 

Thrombocytes Smallest cells in the blood. Normal range 130,000-400,000. Active role in coagulation and hemostasis.

Routine Hematology : 

Routine Hematology Anticoagulant of choice: EDTA Complete Blood Counts (CBCs) Manual WBC Differentials Erythrocyte Sedimentation Rates (ESRs) Sickle Screens Reticulocyte Counts

Automated Counting : 

Automated Counting Coulter Principle Electrical impedance: resistance or change in current when cell passes between two electrodes in NaCl solution.

Automated Counting : 

Automated Counting Flow Cytometry Uses lasers to measure both forward and side scatter. Forward scatter measures size. Side scatter measures granularity.

Sources of Error : 

Sources of Error Inadequate mixing of specimen. Hemolyzed specimens. Lipemic specimens. Cold agglutinins. Clotted specimens. Platelet clumps or platelet satellitosis. Diluted specimens.

Know Normal Ranges!!! : 


Erythrocytic Maturation Series : 

Erythrocytic Maturation Series Rubriblast Prorubricyte Rubricyte Metarubricyte Reticulocyte Erythrocyte

RBC Morphology : 

RBC Morphology

Elliptocytes Target Cells : 

Elliptocytes Target Cells

Tear Drops Stomatocytes : 

Tear Drops Stomatocytes

Sickle Cells Schistocytes : 

Sickle Cells Schistocytes

RBC Inclusions : 

RBC Inclusions

Howell-Jolly Bodies : 

Howell-Jolly Bodies Round, purple inclusions in RBCs. Composed of DNA. Commonly seen in in patients with hypofunctioning spleens. Splenectomy.

Basophilic Stippling : 

Basophilic Stippling Numerous, small purple inclusions in RBCs. Aggregates of ribosomal RNA. Most commonly seen in lead poisoning.

Pappenheimer Bodies : 

Pappenheimer Bodies Clusters of dark blue granules, irregular in size and shape. Composed of iron and ribosomal RNA. Seen in sideroblastic and hemolytic anemias.

Classifications of Anemias : 

Classifications of Anemias Microcytic, Hypochromic Iron deficiency Sideroblastic Chronic disease, Inflammation Lead poisoning Thalassemia trait

Microcytic, Hypochromic : 

Microcytic, Hypochromic Many RBCs smaller than nucleus of normal lymphocytes, increased central pallor. Iron deficiency, thalassemias, anemia of chronic disease.

Classifications of Anemias : 

Classifications of Anemias Normochromic Hereditary Spherocytosis Hereditary Elliptocytosis PNH G6PD deficiency Aplastic anemia Acute blood loss

Classifications of Anemias : 

Classifications of Anemias Macrocytic Vitamin B12 deficiency Folate deficiency Liver disease

Macrocytic RBCs : 

Macrocytic RBCs Most RBCs larger than nucleus of normal lymphocytes, increased MCV. Folate or Vitamin B12 deficiencies, alcoholism, and liver disease.

Reticulocytes : 

Reticulocytes Immature RBCs. Contain residual ribosomal RNA. Reticulum stains blue using a supravital stain (new methylene blue). Counted and expressed as % of total red cells.

Reticulocyte Count : 

Reticulocyte Count Uses supravital stain which stains cells in the living state. Retic % = # retics per 1000 RBCs 10 Corrected retic= % retics x pt. HCT 45

Hemoglobinopathies : 

Hemoglobinopathies Beta Chain Substitutions Hgb S: Valine for glutamic acid (6th position, beta chain) Hgb C: Lysine for glutamic acid (6th position, beta chain)

Hemoglobinopathies : 

Hemoglobinopathies Alkaline Electrophoresis - C S F A + A2 D E G O

WBCEvaluation : 


MyelocyticMaturation Series : 

MyelocyticMaturation Series Myeloblast Promyelocyte Myelocyte Metamyelocyte Band Neutrophil Segmented Neutrophil

Toxic Granulation : 

Toxic Granulation Increased basophilic granules in neutrophils. Seen in severe infections, burns, malignancies, and pregnancy. Distinguish from basophils.

Dohle Bodies : 

Dohle Bodies Sky blue inclusions in cytoplasm of neutrophils. Seen in infections, burns, myleproliferative disorders, and pregnancy. Composed of RER and glycogen granules.

Pelger-Huet Anomaly : 

Pelger-Huet Anomaly

Special Stains : 

Special Stains PAS: ALL, Erytrholeukemia LAP: CML v. Leukomoid Reaction Myeloperoxidase: AML, AMMoL (weak+) Sudan Black: AML, AMMoL (weak+) Specific Esterase: AML, AMMoL Non-specific Esterase: AMML, AMoL TRAP: Hairy Cell Leukemia TDT: ALL Prussian Blue: Sideroblastic Anemia

CML versusLeukomoid Reaction : 

CML versusLeukomoid Reaction Characteristic CML Leukomoid LAP score Decreased Increased Toxic gran. Decreased Increased Dohle bodies Absent Present Philadelphia May be present Absent chromosome

WBC Disorders : 

WBC Disorders Alder-Reilly: Large azurophilic granules, increased mucopolysaccharides Chediak-Higashi: Membrane defect of lysosomes, large primary granules in segs May-Hegglin: Familial disorder with large platelets and Dohle bodies Pelger-Huet: Hyposegmented neutrophils

FAB Classifications : 

FAB Classifications M1: Myeloblastic without maturation M2: Myeloblastic with maturation M3: Promyelocytic M4: Myelomonocytic M5a: Monocytic, poorly differentiated M5b: Monocytic, well differentiated M6: Erythroleukemia, DiGuglielmo’s M7: Megakaryoblastic

Acute Myeblastic Leukemia (M1) : 

Acute Myeblastic Leukemia (M1)

Chronic MyelogenousLeukemia : 

Chronic MyelogenousLeukemia

FAB Classifications : 

FAB Classifications L1: Small, uniform lymphoblasts L2: Large, pleomorhphic lymphoblasts L3: Burkitt’s type (vacuolated and deeply basophilic cytoplasm)

Acute Lymphoblastic Leukemia : 

Acute Lymphoblastic Leukemia

Chronic Lymphocytic Leukemia : 

Chronic Lymphocytic Leukemia

Body Fluids : 

Body Fluids

Types of Body Fluids : 

Types of Body Fluids Cerebrospinal Fluid (CSF) Pleural Fluid Pericardial Fluid Peritoneal Fluid Synovial Fluid Amniotic Fluid Miscellaneous Fluids

Body Fluids Testing : 

Body Fluids Testing Physical examination RBC and WBC counts WBC differential Crystal examination* Rope’s test* *Refers to synovial fluids only.

Cerebrospinal Fluid (CSF) : 

Cerebrospinal Fluid (CSF) Examined to determine the presence of meningitis or intracranial hemorrhage. Multiple tubes drawn (4). Differentiate between traumatic tap and intracranial hemorrhage. Xanthochromia is indicative of intracranial hemorrhage.

Quick Review : 

Quick Review Differentiate between traumatic tap and intracranial hemorrhage. Differentiate between transudate and exudate. Crystal identification and associated conditions.

Traumatic Tap v. Intracranial Hemorrhage : 

Traumatic Tap v. Intracranial Hemorrhage Traumatic Tap Decreasing amounts of blood with each tube drawn May contain clots No xanthochromia Supernatant clear Intracranial Hemorrhage Blood evenly distributed throughout collection tubes No clots Xanthochromia Hemosiderin, hematoidin crystals

Transudates v. Exudates : 

Transudates v. Exudates Characteristic Transudate Exudate Appearance Clear, colorless Yellow, turbid, purulent, bloody Specific gravity <1.015 >1.015 Protein <3 g/dL >3 g/dL LD <200 IU >200 IU Cell count <1000/uL >1000/uL Conditions Congestive Infections, Heart failure Malignancies

Crystal Identification : 

Crystal Identification Monosodium urate (uric acid) Yellow when parallel to the compensator, and blue when perpendicular. Seen in gout. Calcium pyrophosphate Blue when parallel to the compensator, and yellow when perpendicular. Seen in pseudogout.

Crystal Identification : 

Crystal Identification

Good Luck!!!! : 

Good Luck!!!!

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