Practical hematology

Tubes and anticoagulants

 EDTA (Ethylene Diamine Tetra-Acetate)
 Mechanism: forming Ca salts to remove Ca.
 Uses: CBC, PCR, blood films and HbA1c.

 Sodium citrate:
 Anticoagulant: 3.2%
 Mechanism: Calcium chelation.
 Use: Coagulation studies and platelet function.
Black:
 3.8% of sodium citrate
 Action: Remove calcium.
 Uses: Erythrocyte Sedimentation Rate (ESR).
Heparin:
 Sodium Heparin or Lithium Heparin
 Mechanism: inactivation of thrombin and thromboplastin.
 Uses: Use for osmatic fragility test and blood gases

Note: avoid using hemolyzed, clotted or lipemic sample!!!!

Changes in Blood at Room Temperature
 Blood Cell Enumeration-Automated Methods
A. Electrical impedance (coulter): counting & sizing.
• Cells pass through an aperture with an electrical current flowing through
simultaneously. Cells do not conduct current (nonconductive) but rather they
change electrical resistance, which is then counted as voltage pulses.
• The number of pulses generated is proportional to the number of cells present;
amplitude of the pulse generated is proportional to the size of the cell.
• Sample is diluted in isotonic conductive solution that preserves cell shape and
characteristics.
• Dilutions used are dependent on instrument/methodology used.
• Platelets are counted with RBCs.
• Sample for counting WBCs is mixed with reagent to lyse RBCs.
• A commercially available reagent, which both lyses RBCs and converts
hemoglobin to cyanmethemoglobin, can be used to determine hemoglobin and
WBCs in one dilution.
• Hydrodynamic focusing is utilized to reduce cell coincidence (chance of one
cell being counted more than once).
2. Flow cytometry: Optical light scattering. Applications:
• Cell counting, sizing, sorting & WBCs differential. • Immunophenotyping: Differentiating cells on basis
of surface & cytoplasmic markers. Can determine
• Based on both physical and immunologic features. lineage & maturity of cells in hematologic
• Using laminar flow technology (Hydrodynamic focusing), by using malignancies. Useful for Dx, follow-up, &
sheath fluid (normal saline). prognosis.
• Evaluate the size and volume using the forward scatter (FSC) of the • Dx & monitoring of immunodeficiencies
laser beam. • Dx of paroxysmal nocturnal hemoglobinuria
• Enumeration of stem cells
• Evaluate the internal structures (complexity) using the side scatter (SSC) • Quantitation of fetal hemoglobin
of the laser beam.
• Specimens analyzed by flow cytometry: Leukocytes, erythrocytes, lymph
nodes, peripheral whole blood, bone marrow, tumors
• Light source (tungsten halogen lamp or laser light).
• Cells are labeled with one to several fluorescent compounds (excitation
& light emission).
• Energy is detected by a photomultiplier tube and converted into
computerized data, which upon analysis provides information regarding
number, size, and cellular composition of the population assayed.
• The flow cytometer is composed of three main systems: (fluidics, optics
and electronics).
 Immunophenotyping of ALL
A. CD markers of B cell lineage:
• CD10 (CALLA), CD19, CD20, CD34, and TdT (terminal deoxynucleotidyl transferase)
positive.
B. CD markers of T cell lineage:
• CD2, CD3, CD5, CD7 and TdT.
• Pan B cell marker: CD19.
• Pan T cell marker: CD7.
• T helper cell marker: CD4.
• Cytotoxic T cell marker: CD8.

• Immature T cell: CD2 & TdT.

• Immature B cell: CD10, CD19, CD34 & TdT.
+ CD38 (myeloblasts)

(TdT)

Hairy cell leukemia: CD123, CD24, CD11c,

CD103, CD19.
 Automated cell count errors

• WBC counts exceeding instrument linearity limits result in increased cell turbidity and may falsely increase
the hemoglobin, MCH, and MCHC.
• Glucose over 600 mg/dL (hyperosmolarity) may increase the MCV and hematocrit and decrease the
MCHC.
• Cold agglutinins increase the MCV, MCH, and MCHC and decrease the RBC count and hematocrit.
• Lipemia increases the hemoglobin, MCH, and MCHC.
• Repeat the analysis if:
• Rule of three failure on a normocytic sample, especially MCHC >37 g/dL.
• RBC X 3 = Hgb
• RBC X 9=Hct
• Hgb X 3 = Hct
 2-20 fL

Platelet aggregation, clumping,

microcytic RBCs
RBCs histogram
 Hematological tests
1. Erythrocyte Sedimentation Rate:
 ESR measures degree of settling of RBCs in plasma in an anticoagulated specimen during a specific time,
usually 1 hour.
 High fibrinogen or protein levels increase the ESR.
 ESR is increased in chronic inflammatory conditions, including rheumatoid arthritis and pregnancy (increased
fibrinogen), bacterial infection, malignancy, tissue damage, multiple myeloma, Waldenstrom
macroglobulinemia, and severe anemia.
 ESR is normal to decreased in polycythemia, sickle cell anemia, spherocytosis, and other conditions with
poikilocytosis (prevents rouleaux formation).
 Sources of error causing falsely increased results: Tilted column, hemolysis, increased room temperature.
 Sources of error causing falsely decreased results: Clotted sample, excess anticoagulant, "old" blood
(spherocytes form), decreased room temperature, vibration of tube & air bubbles in the tube.
2. Solubility Test for Hemoglobin S:
 Hemoglobin S is insoluble when combined with a reducing agent (sodium dithionite).
 Hgb S will crystallize and give a turbid appearance to the solution.
 The test will not differentiate homozygous from heterozygous conditions containing Hgb S.
 Follow up a positive solubility test with hemoglobin electrophoresis.
3. Hemoglobin Electrophoresis:
 Procedure for the identification of normal and abnormal hemoglobins
 Methodology is based on net negative charges, which cause hemoglobins to migrate from the negative
(cathode) region toward the positive (anode) region. The distance a particular hemoglobin molecule migrates
is due to its net electrical charge.
 Two types of electrophoresis: Cellulose acetate at pH 8.6 and citrate agar at pH 6.2
 Migration of hemoglobin is dependent on net negative charge and buffer pH.
Hematological
calculations
RPI = (Hct / 45) * Retic / Maturation
Atlas
 Basophilic
erythroblast
Polychromatophilic
Pronormoblast erythroblast

Polychromatic cell
(reticulocyte)

Orthochromatophilic Mature
erythroblast erythrocytes
 Mature platelets
Megakaryoblast
Promegakaryocyte Megakaryocyte
Myeloblast Band cell
Promyelocyte Metamyelocyte
Myelocyte
 Mature
monocyte
Monoblast Promonocyte
Macrophage
 Small
lymphocyte

Lymphoblast
Prolymphocyte

Large granular lymphocyte (LGL) or NK cell

Plasma cell
Hint: Compare the small lymphocyte nucleus with
RBCs

Microcytic cell

Macrocytic cell
Normocytic cell
 Polychromatic cells

Anisocytosis (high RDW) Hypochromic cells

Normal RBCs
 Bite cell

Blister cell
Acanthocytes Target cell or Tear drop cell
codocyte

Sickle cell
Burr cell
Schistocyte or
(echinocytes)
helmet cell

Stomatocyte Spherocytes
Rouleaux formation Cold agglutination
(coining) in MM disease (IgM)
Red cell inclusions and parasites:
1. Basophilic stippling: dark purple color, (RNA),
seen in thalassemia, lead poisoning.
2. Cabot ring: oval or eight ring, nuclear membrane
remnants, seen in: lead poisoning, megaloblastic
anemia.
3. Heinze bodies: denatured Hb, seen in: G6PD
deficiency.
4. Hemoglobin C: rod shape crystal, seen in: HbC
disease, HbSC disease.
5. HbH: golf ball like, seen in: HbH disease, alpha
thalassemia.
6. Howell-jolly bodies: nuclear remnants of DNA,
hemplytic anemia, megaloblastic anemia,
thalassemia, sickle cell anemia.
7. Pappenheimer bodies: iron after ineffective
erythropoiesis.
 IDA Beta-thalassemia minor Beta-thalassemia major Megaloblastic anemia

Immune hemolytic anemia HDN Hereditary spherocytosis Hereditary elliptocytosis

MAHA Sickle cell anemia Hb-C disease Hb-SC disease
 Toxic granulation
Pelger-huet anomaly (hyposegmented neutrophils) with dhole body
Dhole body

Hypersegmented neutrophil Vacuolated neutrophil

Normal neutrophil
Reactive lymphocytes
 Acute myeloid leukemia
Myeloblast
Myeloblast Myeloblast

Promyelocytes Myeloblast
Chronic myeloid
leukemia
Acute lymphoblastic leukemia
Chronic lymphocytic leukemia
Phagocytosed
fungi (yeast)
Phagocytosed
histoplasma

Trypanosoma Trypanosoma
brucei cruzi
Anaplasma-Ehrlichia Borrelia Toxoplasma gondii Leishmania
 Leishmaniasis (Amastigotes)
inside macrophage

Microfilaria

Babesia
Ring stages in thick film
Malaria
Alder Reilly anomaly May Hegglin anomaly Chediak-higashi syndrome

Mast cell Thrombocytosis Platelets satellitism

 Lymphocyte VS myelocyte

Monocyte VS large lymphocyte

Reticulocyte stained by new methylene blue Reticulocyte stained by brilliant cresyl blue

Platelets aggregation
(clumping)
This has been a presentation of Alyazeed Hussein
Thanks for your attention and kind patience

@Alyazeed7ussein

@elyazeed7

Alyazeed Hussein, BSc, SUST