Complete Blood Count (CBC)

Complete Blood Count (CBC) Panel of tests that examine different components of the blood. CBC values RBC count Hemoglobin Hematocrit RBC indices WBC count and differential Platelet count

White Blood Count (WBC): actual number of white blood cells per volume of blood. WBC differential: types of WBC present. Red Blood Cells (RBC): actual number of red blood cells per volume of blood Hemoglobin (Hb): amount of the oxygen carrying protein in the blood Platelets (PLT): actual number of platelets per volume of blood

Mean Corpuscular Volume (MCV ): a measurement of the average size of RBCs Mean Corpuscular Hemoglobin (MCH ): the average amount of oxygen-carrying hemoglobin inside a RBC Mean Corpuscular Hemoglobin Concentration (MCHC): the average concentration of hemoglobin inside a RBC Red Cell Distribution Width (RDW): a variation in the size of RBCs

The significance of CBC Find the cause of symptoms such as fatigue, weakness, fever, bruising, or weight loss Diagnosis of anemia Estimation of blood loss Diagnosis of polycythemia Find an infection Diagnosis of blood diseases as leukemia Response to drug or radiation treatment Screening before surgery Abnormal count of certain types of cells

Principle The counting of the cellular elements of the blood (erythrocytes, leukocytes, and platelets) is based on the classic method of electrical impedance. Electrical resistant principle, which depend on the fact that blood cells are non conductive to electricity, so when they pass through electrical field they will increase the electrical resistance. The counting chamber consists of a beaker, two electrodes with a direct current, an orifice with specified dimension; when suspended cells passes through the aperture it will increase the electrical impedance between the two electrodes, manifested as a pulse (sum of pulse= count). The pulse height indicate cell volume.

Performance The aspirated whole blood specimen is divided into two aliquots and mixed with an isotonic diluent. The first dilution is delivered to the RBC aperture bath, and the second is delivered to the WBC aperture bath. In the RBC chamber, both the RBCs and the platelets are counted and discriminated by electrical impedance Particles between 2 and 20 fL are counted as platelets, and those greater than 36 fL are counted as RBCs. Red cell histograms: histograms are derived by plotting the size of each red cell on x axis and the relative number on the y axis. They are used to determine the average size, distribution of size, and to detect sub populations.

Histograms

Hb measurement A reagent to lyse RBCs and release hemoglobin is added to the WBC dilution before the WBCs are counted by impedance After the counting cycles are complete, the WBC dilution is passed to the hemoglobinometer for hemoglobin determination (light transmittance read at a wavelength of 535 nm). Hemoglobin, on most automated systems, is measured as cyanmethemoglobin. Red cells are lysed and potassium ferricyanide oxidizes hemoglobin to methemoglobin, which combines with potassium cyanide forming cyanmethemoglobin. The brown color is measured spectrophotometrically and the corresponding hemoglobin reported. Normal rang Males 14-18 g/dl Females 12-16 g/dl

Normal blood contains about 15-16 grams hemoglobin per 100 ml (dL). Each gram of hemoglobin can carry about 1.35 ml of gaseous oxygen. Fully saturated arterial blood will therefore contain about 20 ml of oxygen per 100 cc. The relative amount of oxygen in the blood compared to the carrying capacity of the hemoglobin is called the oxygen saturation, and is expressed as a percentage.

Hematocrit Hematocrit is the volume of the red cells as compared to the volume of the whole blood sample. Hematocrits on the automated systems are calculated. Calculated: (MCV)×(RBC) = Hct Usually expressed in percentage (42%) Males 42-52 % Females 37-47% (pregnant>33%)

Provides information on the amount of red blood cells (RBC) present in the blood. Decreased levels means anemia from hemorrhage, parasites, nutritional deficiencies or chronic disease process, such as liver disease, cancer, etc. Increased levels are often seen in dehydration or polycythemia

Red Blood Cell Count (RBC) Normal value = 4.6 to 6.2 x 106 cells/L Decreased with anemia Increased with erythrocytotic states such as polycythemia vera, erythrocytosis of chronic hypoxia, dehydration, stress polycythemia, and thalassemia minor.

MCV Mean cell volume MCV is average size of RBC MCV = Hct x 10 RBC (millions) If 80-100 fL, normal range, RBCs considered Normocytic If < 80 fL are Microcytic If > 100 fL are Macrocytic Not reliable when have marked anisocytosis

MCH MCH is average weight of hemoglobin per RBC. MCH = Hgb x 10 RBC (millions)

MCHC MCHC is average hemoglobin concentration per RBC MCHC = Hgb x 100 Hct (%) If MCHC is normal, cell described as Normochromic If MCHC is less than normal, cell described as Hypochromic There are no Hyperchromic RBCs

RDW An index of RBC size variation May be used to quantitate the amount of anisocytosis on peripheral blood smear Normal range is 11.5% to 14.5% for both men and women. MPV: The MPV is a measure of the average volume of platelets in a sample and is analogous to the erythrocytic MCV. Pct : analogues to HCT for RBCs

Use of RBC indices in differential diagnosis can provide picture of what is occurring clinically. If anemia caused be bone marrow failure, requires information about RBC production. Information obtained from reticulocyte count. Reticulocyte count measures effective RBC production. As study different anemias, will learn morphology.

Red Cells Histogram Noramal red cell histogrem dispays cells form (36- 360 ) fl (24- 36 fl ) flag may be due 1- RBCs fragments 2- WBCs fragments 3- Giant plts 4- Microcyte

Shift to right : - Leukemia - Macrocytic anemia - Megaloblastic anemia Shift to left : - Microcyic anemia (IDA) Bimodal - Cold agglutinin - IDA, megaloblastic anemia with transfusion. -Saidroblastic anemia. Trimodal Anemia with transfusion

Platelet Count Normal Range = 150 to 450 x 103 cells/L Thrombocytosis inflammatory disorders myeloproliferative states acute blood loss hemolytic anemias carcinomatosis status post-splenectomy exercise etc.

Thrombocytopenia Production defects such as aplastic anemia, marrow replacement, megaloblastic and severe iron deficiency anemias, uremia etc. Consumption defects with autoimmune thrombocytopenias, DIC, hypersplenism, massive hemorrhage and many severe infections.

Plts histogram Normal Rang (2-20 fl) (0-2) 1- Air Babbles 2- Dust Over 20 fl 1- Microcyte 2- Scishtocyte 3- WBCs fragments 4- Giant Plts 5- Clumped plts

Reticulocytes Useful in determining response and potential of bone marrow. Reticulocytes are non-nucleated RBCs that still contain RNA. Visualized by staining with supravital dyes, including new methylene blue; RNA is precipitated as dye-protein complex. Normal range is 0.5-2.0% of all erythrocytes. If bone marrow responding to anemia, should see increases in Retic count. Newborns have higher Retic count than adults until second or third week of life.

Reticulocytes

Sickle cells (Dreprnocytes)

A variation in erythrocyte distribution such as rouleaux formation or agglutination

White Blood Cell Count (WBC) The white blood cell differential count determines the number of each type of white blood cell, present in the blood.  It can be expressed as a percentage (relative numbers of each type of WBC in relationship to the total WBC) or as an absolute value (percentage x total WBC). Of these, the absolute value is much more important than the relative value. There are five basic white blood cell types: Neutrophils Eosinophils Basophils Lymphocytes Monocytes Each WBC cell type has its' own unique features.

Neutrophils Polymorphonuclear Neutrophils These are the most common of the WBCs and serve as the primary defense against infection. The typical response to infection or serious injury is an increased production of neutrophils.

Bands/Stabs Early in the response to infection, immature forms of neutrophils will be seen. These are call Stab or Band cells. The presence of these immature cells is called a "shift to the left" and can be the earliest sign of a WBC response, even before the WBC becomes elevated.

Eosinophils These cells play a role in allergic disorders and in combating parasitic infections. Elevations in eosinophil counts are associated with: Allergic reactions Parasite infections Chronic skin infections Some cancers Decreases in eosinophil counts are associated with: Stress Steroid exposure Anything that may suppress WBC production generally

Basophils These cells can digest bacteria and other foreign bodies (phagocytosis) and also have some role in allergic reactions. Elevations in basophil counts are associated with: Some cancers Some allergic reactions Some infections Radiation exposure Diminished basophil counts are associated with: Stress reactions Hyperthyroidism Prolonged steroid expo

Monocytes These cells respond to inflammation, infection and foreign bodies by ingesting and digesting the foreign material. Increased monocyte counts are associated with: Recovery from an acute infection Viral illness Parasitic infections Collagen disease Some cancers Decreased monocyte counts are associated with: HIV infection Rheumatoid arthritis Steroid exposure

Lymphocytes These cells play both an immediate and delayed role in response to infection or inflammation. Increased numbers of lymphocytes are seen in: Most viral infections Some bacterial infections Some cancers Graves' disease Decreased numbers of lymphocytes are seen in: Steroid exposure Immunodeficiency Renal failure Lupus

White Blood Cell Count (WBC) The lysing reagent also cause WBCs membrane collapse aruond the nucleus , so the counter actually measuring the nuclear size. Cells lies between (35-90 fl) are considered lymphocyte. (90-160 fl) are considered MID cells (160-450 fl) are neutrophile.

Anemia

Definition of Anemia Inability of blood to supply tissues with adequate oxygen for proper metabolic function. Usually associated with decreased levels of hemoglobin or hematocrit (packed red cell volume) Usually associated with decreased RBCs. Diagnosis made by patient history, physical examination, signs and symptoms, and hematological laboratory findings. Classified as moderate (Hb 7-10 g/dl) or severe (Hb <7g/dl). Two general forms of anemia:   Absolute Anemia (decrease in red cell mass) and Relative Anemia (increased plasma volume gives appearance of anemia).

RBC and Hemoglobin Production In healthy individuals, about 1% of RBCs lost daily. Bone marrow continuously produces RBCs to equal daily loss. Reticulocyte count is a lab measurement of this loss. Normal Retic count is 0.5-2.0% of circulating RBCs. Replacement requires functioning bone marrow, normal RBC maturation and ability to release mature RBCs to peripheral blood. Proper nutrition required (B12, Folate).  Also requires normal hemoglobin synthesis.

Erythropoietin levels (Epo) useful diagnostic tool. Erythropoietin is a hormone produced in the kidney. Levels of erythropoietin varies with oxygen tension in kidney tissues (↓ Oxygen -↑ Epo, and vice versa). Anemic people usually respond by increasing erythropoietin levels. Clinical Diagnosis Made by combination of factors including: patient history, physical signs and changes in hematologic profile (CBC). Signs and symptoms usually non-specific: fatigue, weakness, shortness of breath - especially after exertion.

Functional classification of Anemias Decreased RBC production (hypoproliferative) Defective hemoglobin synthesis Fe deficiency B12 deficiency Folate deficiency Impaired bone marrow or stem cell function, as in leukemia Increased RBC destruction, as in sickle cell anemia or hemolytic anemia Combination of the two (sometimes called “ineffective erythropoiesis”)

Decreased MCV and Decreased MCHC Microcytic/ Hypochromic Anemia Iron deficiency Thalassemia Anemia of chronic disease Sideroblastic anemia Lead poisoning Increased MCV, Decreased MCHC Macrocytic/ Normochromic Anemia Folate deficiency B12 deficiency Hypothyroidism

Iron Deficiency Anemia Iron metabolism Absorption in duodenum Transferrin transports iron to the cells. Ferritin and hemosydrin store iron. 10% of daily iron is absorbed Most body iron is present in hemoglobin in circulating red cells The macrophages of the reticuloendotelial system store iron released from hemoglobin as ferritin and hemosiderin Small loss of iron each day in urine, faeces, skin and nails and in menstruating females as blood (1-2 mg daily)

Thalassemias

Introduction Heritable, hypochromic anemias-varying degrees of severity Genetic defects result in decreased or absent production of mRNA and globin chain synthesis At least 100 distinct mutations High incidence in Asia, Africa, Mideast.

Globin Chains Alpha Globin 141 amino acids Coded for on Chromosome 16 Found in normal adult hemoglobin, A1 and A2 Beta Globin 146 amino acids Coded for on Chromosome 11, found in Hgb A1 Delta Globin Found in Hemoglobin A2--small amounts in all adults Gamma Globin Found in Fetal Hemoglobin Zeta Globin Found in embryonic hemoglobin

Hemoglobin Types Globin Chains Hemoglobin Type a2b2 a2d2 a2g2 b4 g4 a2b26 gluval a2b26 glulys Hemoglobin Type Hgb A1—92%--------- Hgb A2—2.5%-------- Hgb F — <1%--------- Hgb H ------------------ Bart’s Hgb-------------- Hgb S-------------------- Hgb C-------------------

Alpha Thalassemias Result from gene deletions One deletion: Silent carrier; no clinical significance Two deletions: a Thal trait; mild hypochromic microcytic anemia Three deletions: Hgb H; variable severity, but less severe than Beta Thal Major Four deletions: Bart’s Hgb; Usually no treatment indicated 4 deletions incompatible with life 3 or fewer deletions have only mild anemia

Beta Thalassemias Result from Point Mutations on genes b0-no b-globin synthesis; b+ reduced synthesis Disease results in an overproduction of a-globin chains, which precipitate in the cells and cause splenic sequestration of RBCs Erythropoiesis increases, sometimes becomes extramedullary