Basic Hematology PDF

Basic Hematology LEARNING OBJECTIVES 1. Define "CBC" 2. Define and explain red cell indices including *derived values* such as MCV, MCHC and MCH 3. Define and explain erythrocyte sedimentation rate 4. Describe the main cell types observed in peripheral blood. 5. Describe what is meant...

Basic Hematology LEARNING OBJECTIVES 1. Define "CBC" 2. Define and explain red cell indices including *derived values* such as MCV, MCHC and MCH 3. Define and explain erythrocyte sedimentation rate 4. Describe the main cell types observed in peripheral blood. 5. Describe what is meant by the term "differential count". 6. Describe the distribution & morphology of cells in certain common "Blood Pictures" [Clinical Diagnostic Laboratories offer]: 1. 2. 3. What do these terms mean, and how are they used? 1. **White Cell count (leukocyte count)** 2. **Platelet count** 3. **Red cell count, hemoglobin concentration and red cell indices** What Specimen? - - White cells, red cells (erythrocytes) and platelets are counted **per unit volume** of whole blood. - Unit volume: per **[cubic millimeter]** (mm^3^) which is the same as **µL** - **WBC**4.0-10.0 x 103/cu mm - **Platelets** 150-450 x 103/cu mm - **RBC**4.5-5.9 x 106/cu mm - Unit volume: per **liter (SI units)**. - **WBC**4.0-10.0 x 109/L - **Platelets** 150-450 x 109/L - **RBC**4.5-5.9 x 1012/L 1. Hemoglobin 2. Hematocrit 3. Red cell count 4. Mean cell (corpuscular) *volume* or MCV 5. Mean cell *hemoglobin concentration* or 6. Mean cell *hemoglobin content* or MCH 7. Red cell distribution width or RDW 1. **[Hemoglobin concentration]** in grams/dL (grams per 100 mL) - **Hemoglobin is converted to CYANMETHEMOGLOBIN which absorbs maximally at 540 nm** - Hb plus [KCN] plus [potassium ferricyanide] + [NaHCO]~3~ - Heme iron (Fe2+) → Fe3+ - Methemoglobin binds with **CN** - **Most current hematology analyzers have switched to a new, alternative method: *[sodium lauryl sulfate]* (SLS, SDS) binds to hemoglobin.** - **Sulfated derivative absorbs at 535 nm** ---------------- **Absorbance** ---------------- OXYHEMOGLOBIN **CYANMETHEMOGLOBIN** ---------------- **Absorbance** ---------------- **Na Lauryl Sulfate (SLS)-Hemoglobin**  **CYANMETHEMOGLOBIN** Hematocrits determined by blood centrifugation are termed "*spun* *hematocrits*" *Normal: 35 - 45% vol/vol* 2. **[Hematocrit]** (the percentage of blood that is represented by the packed red cells)  3. [**Red cell count**.] *Number* of red blood cells per microliter of blood (or per liter) 4. High MCV = Macrocytic Normal: 78 -100 *femtoliters* 5. **[MCHC]** ([M]ean [c]ell [h]emoglobin [c]oncentration: Hemoglobin concentration of the packed red cells (minus plasma) 6. **[Mean Cell Hemoglobin]** (**MCH**) = red cell hemoglobin **content** in picograms or 10-12 grams **MCV**: reflects the Cell *[Volume]* in femtoliters **MCH**: reflects the Hb *[CONTENT]* (in Both picograms) of each red cell  **[femtoliters]** **[g/dL of erythrocytes]** 7. - The *coefficient of* *variation* of the red cell volume - distribution histogram MCV 1. Directly measured  1. 2. - **In a hemolytic anemia → ↑ reticulocytes** - **In bone marrow disease (impaired erythrocyte production) → ↓ reticulocytes** Normal stain Using special reticulocyte (supravital) stain  - -- -- -- -- -- -- -- -- Erythrocyte sedimentation rate or ESR - Anticoagulated whole blood (4 parts whole blood: 1 part Na Citrate) is placed in a narrow-bore glass tube tube. Erythocytes are allowed to sediment at room temperature. - ESR measures the distance, in Sedimenting ------------- -- RBCs  Erythrocyte sedimentation rate or ESR - - - VERY LOW SPECIFICITY Erythrocyte sedimentation rate or ESR - [Westergren Method]: 200 mm tube - [Wintrobe Method]: 100 mm tube - [Modern Methods]: Semi-automated systems that measure sedimentation by infra-red light. Measured over a shorter period than 1 hour & extrapolated to give Westergren ESR. - Can interface with LIS  The Blood Smear  Stained Blood Smear Neutrophil ------------ -- -- -- Monocyte Basophil MYELOID 1. Stained peripheral blood smear is examined carefully using 40 x to 100 x objective -- usually oil immersion lens 2. 100 white blood cells are counted 3. Cells are classified by morphology→ Neutrophils Bands Lymphocytes Monocytes Eosinophils Basophils Metamyelocytes Myelocytes Promyelocytes Blasts  Eosinophil, Lymphocyte & Neutrophil     **Basophils** - involved in IgE-mediated hypersensitivity response Promyelocyte ^Metamyelocyte^ Band segmented Cell   Myelocytes - very immature neutrophils with a round or near-round nucleus         END Regarding the *[m]ean* *[c]ell* *[v]olume* or MCV: a. b. c. d. e. Explanation: The red cell MCV is measured directly on cell counters; this is true whatever method is employed to enumerate cells. The counter is able to plot a red cell volume histogram, and the mean is determined. MCV can be calculated from the spun hematocrit as in option b. This was the original method for determining MCV but it is not commonly used anymore. The Erythrocyte Sedimentation Rate (ESR) is: a. b. c. d. e. Explanation: The ESR is the rate of sedimentation of red cells in a whole blood specimen enclosed in a narrow-bore glass tube. ESR is increased in acute inflammation. Self-Assessment Question \#3 Acute leukemia is characterized by presence of \_\_\_\_ in the stained peripheral blood smear: a. b. c. d. e. Explanation: The hallmark of acute leukemia is the increased presence of blasts in the bone marrow and in peripheral blood. Blasts are immature progenitor cells with characteristic morphology and cell surface markers.  - 0.45 L/L divided by 5.0 x 10^12^/L - = 0.09 x 10^-12^ L - = 90 x 10^-15^ L or 100 fL (femtoliter) - A femtoliter (fL) is = "*cubic micron* (µM^3^)" - 150 g/L divided by 0.45 L/L - = 333 g/L - = 33.3 g/dL - 150 g/L divided by 5.0 x 10^12^/L - = 30 x 10^-12^g - = 30 pg  AUTOMATED CELL Analysis Conductivity Differential Responses to Chemical Treatment  Electrically conductive diluent -- -- -- ---- -- -- -- -- -- -- -- ------- -- -- -- -- -- -- -- **+** \- -- -- -- ---- -- -- -- -- -- -- -- ------- -- -- -- -- -- -- --  - Current interruption = "Impedance" - Impedance can be recorded as *voltage* *pulses* - The [number] of pulses = Cell Number - Pulse [height] = *Cell Volume* \[femtoliters\]  **Conductivity** is determined by *cell* *granularity, nuclear density, nuclear/cytoplasm ratio* 3. **LASER** CELL  SIDE SCATTER: Internal complexity 4. Differential Responses to Chemical treatment - Lysis of RBCs → Allows counting of white cells - Selective *shrinking* and/or *lysis* of certain white cell populations - Selective *stripping* of cytoplasm → Allows analysis of cell nuclei - Selective staining of cells e.g., *myeloperoxidase* New Generation Cell Counters - High sensitivity & specificity for blasts - Ability to distinguish lymphoblasts from normal lymphocytes - Automated enumeration of nucleated RBCs with correction of WBC count - Reticulocyte counting, unaffected by red cell shape and capable of providing *immature reticulocyte fraction* and/or CHr - High sensitivity & specificity for *immature granulocytes* - Some will enumerate immature granulocytes - Automated slidemaker/stainer - Can be connected to automated front-end processing