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 CBCThe 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 axisx axis and the relative number on the y axisy 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.

MPVMPV: : The MPV is a measure of the average volume of platelets in a sample and is analogous to the erythrocytic MCV.PctPct : : 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 due1- RBCs fragments2- WBCs fragments 3- Giant plts4- 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.

TrimodalAnemia 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 Babbles2- Dust

Over 20 fl1- Microcyte2- Scishtocyte3- WBCs fragments

4- Giant Plts5- 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.

NeutrophilsPolymorphonuclear 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 Some allergic reactions Hyperthyroidism Prolonged steroid expo

MonocytesThese 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

Some cancers

LymphocytesThese 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 Some cancers 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.

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 ProductionRBC 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 synthesisFe deficiencyB12 deficiencyFolate deficiencyImpaired 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

Macrocytic/ Normochromic AnemiaFolate deficiencyB12 deficiencyHypothyroidism

Increased MCV, Decreased MCHC

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)

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

Hemoglobin Type• Hgb A1—92%---------• Hgb A2—2.5%--------• Hgb F — <1%---------• Hgb H ------------------• Bart’s Hgb--------------• Hgb S--------------------• Hgb C-------------------

Globin Chains a2b2 a2d2 a2g2 b4 g4 a2b26 gluval

a2b26 glulys

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