Complete Blood Count (CBC) PDF

THE COMPLETE BLOOD COUNT The complete blood count (CBC) is the foundation procedure performed in a hematology laboratory. A CBC consists of specific measurements of hemoglobin, hematocrit, red and white blood cell counts, platelet count, leukocyte differential, and evaluation of a peripheral blood s...

THE COMPLETE BLOOD COUNT The complete blood count (CBC) is the foundation procedure performed in a hematology laboratory. A CBC consists of specific measurements of hemoglobin, hematocrit, red and white blood cell counts, platelet count, leukocyte differential, and evaluation of a peripheral blood smear as basic information. These tests are essential to the initial evaluation and follow-up of a patient. Overall Quantitative Measurements Quantitative measurements of erythrocytes, leukocytes and platelets are a standard part of the report generated by automated instrumentation. Although generally replaced by automated cell counts, manual cell counts (see Chapter 32, Manual Procedures) may be needed. In most cases, RBC or WBC counts are only performed manually when there are extremely low total leukocyte counts from whole blood or for counting cells in body fluids, such as cerebrospinal fluid or synovial fluid (see Chapter 29, Body Fluids). Manual determinations of hemoglobin and centrifuge- based measurement of the microhematocrit can be a quality control strategy or as backup methods of analysis. The RBC indices of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) are now a standard part of a routine automated CBC. Through the use of automated hematology instrumentation, additional measurements have been added to a report, including reticulocyte information, red cell distribution width (RDW), and blood cell histograms. Measurement and analysis of cells by automated instrumentation is discussed in Chapter 30. Manual Erythrocyte, Leukocyte, and Platelet Counts In manual cell counting, blood specimens are diluted to an exact ratio with specific diluents (see Table 10.1), and the cells are counted in a hemacytometer, an accurately ruled chamber ruled off in areas of square millimeters (see Figs. 32.1 and 32.3). TABLE 10.1 Manual Total Cell Counts  Description In order to count leukocytes, the diluting fluid must destroy the more numerous RBCs so WBCs may be counted more readily. (WBCs need not be eliminated when RBCs are being counted.) The principle of osmotic pressure is again employed but in a different way. In the classic procedure, a lysing agent hemolyzes the RBCs and converts the hemoglobin released from the red cells into acid hematin, which gives the resulting solution a brown color. The intensity of the brown color is directly related to the amount of hemoglobin present in the RBCs. QUANTITATIVE ASSESSMENT OF ERYTHROCYTES Hemoglobin Measurement in the Laboratory The determination of hemoglobin (Hb) can be performed separately or as part of a routine CBC. A hemoglobin measurement is a standard part of automated instrumentation, but it may be performed manually as well. A manual determination of hemoglobin can be used as a quality control measure or as a backup methods of analysis. The hemiglobincyanide method, HiC, or cyanmethemoglobin method uses a modified Drabkin’s reagent that contains potassium cyanide; potassium ferricyanide; dihydrogen potassium phosphate (KH2PO4), which shortens the conversion time to 3 minutes; and a nonionic detergent that minimizes turbidity and enhances RBC lysis. When the cyanmethemoglobin reagent is mixed with the blood specimen, the stable pigment HiCN is formed and can be measured quantitatively in a spectrophotometer. Hemoglobin determinations done by an automated instrument generally use the traditional cyanmethemoglobin method. The sample is lysed by using the detergent- modified Drabkin’s reagent, and light absorbance is measured at 540 nm. Sources of error in the measurement of hemoglobin can result from specimen conditions. Hemoglobin concentration can be falsely elevated by lipemia (cloudy plasma), extremely elevated WBC count, or red blood cells containing hemoglobin C or hemoglobin S. Strategies for problem-solving conditions that cause false results include the following. If a lipemic specimen is encountered, replace the lipemic plasma with an equal amount of 0.85% saline, mix the specimen, and retest the specimen. In the case of an extremely elevated WBC count, the patient specimen that produces the cloudy hemoglobin solution can be centrifuged, the clear supernatant can be transferred to a cuvette, and the %T of the supernatant fluid can be read on a spectrophotometer. If an error due to hemoglobin C or S is encountered, the blood specimen can be diluted 1:2 with distilled water with the final result being multiplied by 2. Hematocrit (Packed Cell Volume) The hematocrit (Hct), or packed cell volume, is a macroscopic observation of volume of the packed RBCs in a sample of whole blood, if measured by manual technique. The manual procedure is relatively simple and reliable. A hematocrit is used in evaluating and classifying the various types of anemias according to red cell indices. When whole blood is centrifuged, the heavier particles fall to the bottom of the tube, and the lighter particles settle on top of the heavier cells. The hematocrit is the percentage of RBCs in a volume of whole blood. It is expressed as units of percent or as a ratio in the SI system. An automated hematocrit result is obtained when multiparameter instruments are used. This result is computed from individual red cell volumes (MCV) and the red cell count and is not affected by the trapped plasma that is left in the RBC column for the manual methods. Hematocrit value obtained with the automated instruments is slightly lower than the value obtained by the centrifugation methods. Sources of error in manually performed microhematocrit determinations can include specimen or technical errors. Specimen errors can produce falsely decreased results because of an inadequately filled EDTA anticoagulant evacuated tube that causes RBC shrinkage. Technical sources of error include overcentrifugation or improper sealing of the test capillary tube. Patients with red blood cell disorders such as macrocytic anemia or sickle cell anemia can demonstrate a falsely elevated result. Rule of Three A quick check of hemoglobin and microhematocrit results is by the “rule of three.” This rule states that the microhematocrit should be three times the value of the hemoglobin plus or minus 3 percent (Hgb × 3 = HCT ± 3%). Example: If a patient has a hemoglobin of 11.0 g/dL and a microhematocrit of 35%, the rule of three is determined by multiplying 11 times 3 yielding a result of 33. The acceptable range for this result is 30 to 36 (or 30% to 36%). This rule only applies to normochromic, normocytic red blood cells. A discrepancy in the value may indicate abnormal red blood cells or a measurement error. Blood Volume Measurement In most cases, the total number of erythrocytes is closely related to the red cell concentration of the blood or Hct but blood volume may not reflect the erythrocyte concentration in conditions such as immediately after a severe hemorrhage, severe dehydration, or overhydration. To accurately assess the blood volume in these patients, plasma volume or red cell mass or volume must be determined. Plasma volume is measured by dilution methods. A substance that is confined to the intravascular plasma compartment, such as Evans blue dye, 131I-labeled albumin, or radioactive indium-labeled transferrin, is injected and the volume distribution is calculated from the degree of dilution of the injected substance over a period of 15 to 30 minutes. Radiolabeled albumin is the most commonly used, but corrections must be made because the label is gradually removed from the circulation into the extravascular space, leading to errors of 10% or more in plasma volume determinations. Plasma volume may also be estimated from red cell volume using radioactive, labeled red blood cells. The radioisotopes 51Cr and 99mTc are the most commonly used. Red cell volume may also be calculated from the total plasma volume and measured hematocrit. NOTE: This is a good time to review the definition of the Key Terms in the Glossary and flash cards on the Navigate 2 Advantage course. It is also a good time to complete Review Questions related to the preceding content. Manual Erythrocyte Count Red Cell Count Manual methods for counting red cells have proven to be very inaccurate, and automated counters provide a much more accurate reflection of red cell quantities. In the analysis of red blood cells, an isotonic solution is used as the diluent for whole blood. Because the number of red cells greatly exceeds the number of white cells (by a factor of 500 or more), the error introduced by counting red cells and white cells is negligible. However, when marked leukocytosis is present, red cell counts and volume determinations may be erroneous unless corrected for the presence of white cells. The observed precision for RBC counts using automated hematology analyzers is 1% (CV) compared with a minimum estimated precision value of 11% with manual methods. Calculation: No. of RBCs = average total of RBCs in 5 squares × dilution correction factor × volume correction factor 1. 5 of the 25 squares in the large 1-mm square are counted. 2. The specimen dilution factor is 200. 3. The volume correction factor is 50. This number represents the total volume of five squares reported in terms of 1.00 μL. This is calculated by dividing the volume desired (1.00 μL) by the volume used (0.02/μL). Example: If the average number of RBCs counted is 400, the total RBC count is 400 × 200 × 50 = 4.0 × 1012/L A simplification of this formula is to use a factor of 10,000, which represents 200 × 50. The total average of 400 RBCs × the factor of 10,000 = 4,000,000 or 4 × 1012/L. Sources of Error: Increased or erratic results may be seen if contaminated diluting fluid, wet or dirty pipettes, a dirty hemacytometer, or drying of the dilution in the hemacytometer occurs. Reference Ranges: Normal values for erythrocytes and related measurements appear in Table 10.2. TABLE 10.2 Values in the Measurement of Erythrocytes Description SI, Systeme International d’Unites. Source: Perkins SL. Normal blood and bone marrow values in humans. In: Greer JP, et al. (eds.). Wintrobes’ Clinical Hematology, 11th ed, Philadelphia, PA: Lippincott Williams & Wilkins, 2004:2607; Handin RI, Lux SE, Stossel TP. Blood, 2nd ed, Philadelphia, PA: Lippincott Williams & Wilkins, 2003; Appendix 25, Red Blood Cell Values at Various Ages, p. 2216. One femtoliter (fL) = 1015 L = 1 cubic micrometer; One picogram (pg) = 1012 g = 1 micromicrogram. Various patient conditions can produce an increased or decreased concentration of red blood cells. A decrease in RBCs can be encountered in anemias, hemorrhage, hemolytic conditions, and acute leukemias. An increased concentration of RBCs is seen in conditions such as dehydration or polycythemia. Red Blood Cell Indices The erythrocyte indices are used to mathematically define cell size and the concentration of hemoglobin within the cell. They are 1. Mean corpuscular colume (MCV) 2. Mean corpuscular hemoglobin (MCH) 3. Mean corpuscular hemoglobin concentration (MCHC) The MCV, MCH, and MCHC reflect average red blood cell values and may not correctly describe blood specimens when mixed populations of red cells are present. For example, in sideroblastic anemias, a dimorphic red cell population of both hypochromic and normochromic cells may be present but the indices may be normochromic and normocytic. It is important to examine the blood smear as well as red cell histograms to detect such dimorphic populations. The MCV is an extremely useful value in classification of anemias, but the MCH and MCHC may not add significant, clinically relevant information. The MCH and MCHC can have an important role in laboratory quality control because these values will remain stable for a given specimen over time. Mean Corpuscular Volume The MCV expresses the average size or volume of an erythrocyte. The formula is Example: If the patient’s hematocrit is 35%, or 0.35 L/L, and the erythrocyte count is 4.0 × 1012/L, the MCV is determined thus: a One femtoliter (fL) = 10−15 L = 1 cubic micrometer (μm3) Reference range (adult) for MCV is 80 to 96 fL Clinical Conditions: The MCV is a useful parameter for classification of anemias based on size. Elevated MCV values are associated with macrocytic anemias and pathological megaloblastic anemias such as pernicious anemia and folic acid deficiencies. Low MCV values are associated with iron deficiency anemia, heterozygous thalassemias, and anemias of chronic inflammation /anemia of chronic disorders. Sources of Error: Autoagglutination of red cells in conditions such as cold agglutinin disease or paraproteinemia may result in a falsely elevated MCV. Most automated analyzers gate out MCV values above 360 fL, which excludes most red cell clumps, but this may falsely lower Hct values. In addition, severe hyperglycemia (glucose less than 600 mg/dL) may cause osmotic swelling of the red cells, which leads to a falsely elevated MCV. Leukocytosis may also spuriously elevate MCV values. Mean Corpuscular Hemoglobin The MCH expresses the average weight (content) of hemoglobin per red cell. It is directly proportional to the amount of hemoglobin and the size of the erythrocyte. The formula is Example: If the patient’s hemoglobin is 14 g/dL and the erythrocyte count is 4 × 10 /L, the MCH would equal 12 b One picogram (pg) = 10−12 g = 1 micromicrogram (μμg) Reference range (adult) for MCH is 27.5 to 33.2 pg Clinical Conditions: In anemias secondary to impaired hemoglobin synthesis, such as iron deficiency anemia, the hemoglobin mass per red cell decreases, resulting in a lower MCH value. Sources of Error: MCH measurements may be falsely elevated by hyperlipidemia as increased plasma turbidity will erroneously elevate hemoglobin measurement. Centrifugation of the blood sample to eliminate the turbidity followed by manual hemoglobin determination allows correction of the MCH value. Mean Corpuscular Hemoglobin Concentration The MCHC expresses the average concentration of hemoglobin per unit volume of erythrocytes in the sample. It is also defined as the ratio of the weight of hemoglobin to the volume of erythrocytes. The formula is  Example: If the patient’s hemoglobin is 14 g/dL and the hematocrit is 45% or 0.45 L/L, the MCHC would equal: Reference range (adult) for MCHC is 33.4% to 35.6% Clinical Conditions: Fifty percent of patients with hereditary spherocytosis demonstrate an elevated MCHC. This makes spherocytosis the most common condition associated with an increased MCHC. Other specimen conditions that can yield an MCHC over 36 g/dL include conditions of lipemia (abnormally high plasma lipid) or active cold agglutinin disease. cold agglutinin disease. Sources of Error: The accuracy of the MCHC determination is affected by factors that affect measurement of either Hct such as plasma trapping or presence of abnormal red cells or hemoglobin such as hyperlipidemia or leukocytosis. In cases of autoagglutination at room temperature due to a cold agglutinin, the MCV will be falsely elevated and the RBC count will be falsely decreased. These two inaccurate measurements will result in an elevated MCHC. To correct for this type of error, a blood specimen must be warmed to 37°C and the measurements should be repeated Other sources of error in calculating the MCHC include artifactual elevation of hemoglobin concentration in photometric measurements due to lipemia. Hemolysis can also affect the MCHC. With the exception of hereditary spherocytosis and some cases of homozygous sickle cell or hemoglobin C disease, MCHC values will not exceed 37 g/dL. This level is close to the solubility value for hemoglobin. Further increases in Hb may lead to crystallization. Red Cell Distribution Width The red cell distribution width (RDW) is a red cell measurement that quantitates cellular volume heterogeneity reflecting the range of red cell sizes within a sample. RDW has been proposed to be useful in early classification of anemia as it becomes abnormal earlier in nutritional deficiency anemias than do other red cell parameters, especially in cases of iron deficiency anemia RDW is particularly useful in characterizing microcytic anemia, allowing to distinguish between uncomplicated iron deficiency anemia (high RDW, normal to low MCV) and uncomplicated heterozygous thalassemia (normal RDW, low MCV), although other tests are usually required to confirm the diagnosis. RDW is also useful in identifying red cell fragmentation, NOTE: This is a good time to complete Review Questions related to the preceding content. Reticulocytes As an erythrocyte develops, the nucleus becomes more and more condensed and is eventually lost. After the loss of the nucleus, an immature erythrocyte (reticulocyte) remains in the bone marrow for 2 to 3 days before entering the circulating blood. During this period in the bone marrow and during the first day in the circulation, this immature erythrocyte is referred to as a reticulocyte. Although the reticulocyte lacks a nucleus, it contains various organelles, such as mitochondria, and an extensive number of ribosomes. The formation of new ribosomes ceases with the loss of the nucleus in the late metarubricyte; however, while RNA is present, protein and heme synthesis continues. During reticulocyte maturation, the RNA is catabolized, and the ribosomes disintegrate. The loss of ribosomes and mitochondria, along with full hemoglobinization of the cell, marks the transition from the reticulocyte stage to full maturation of the erythrocyte. Under normal conditions, the quantity of reticulocytes in the bone marrow is equal to that of the reticulocytes in the circulating blood. To maintain a stable reticulocyte pool in the circulation, the bone marrow replaces the number of erythrocytes that have reached their full life span. Because the normal life span or survival time is 120 days, 1/120th of the total number of erythrocytes is lost each day, and an equal number of reticulocytes is released into the circulation. If, under the influence of erythropoietin to stimulate RNA synthesis of erythroid cells, increased numbers of young reticulocytes are prematurely released from the bone marrow because of such conditions as acute bleeding, these reticulocytes are referred to as stress or shift reticulocytes. This situation is analogous to the appearance of immature leukocytes in the peripheral blood during the stress of infection. Reticulocyte Count Peripheral smears of normal blood stained with Wright stain may demonstrate a slight blue tint in some erythrocytes. This morphological condition of erythrocytes, which is described in more detail in Chapter 5, is referred to as polychromatophilia or polychromasia.  Supravital staining is important for reticulocyte detection because the red blood cells must be living in order to stain the RNA remaining in the cell. If a supravital stain, such as new methylene blue, is used, it precipitates the ribosomal RNA in these cells to form a deep-blue, meshlike network. Stress reticulocytes are recognizable on Wright-stained blood smears by their larger size and increased blue tint and may be accompanied by even younger erythrocytes, such as metarubricytes. When stained with a supravital stain, stress reticulocytes exhibit a much denser meshlike network. The reticulocyte count procedure is frequently performed in the clinical laboratory as an indicator of the rate of erythrocyte production. Usually, the count is expressed as a percentage of total erythrocytes. The normal reference range is 0.5% to 1.5% in adults. In newborn infants, the range is 2.5% to 6.5%, and 1% to 3% at 2 weeks of age to 1 year of age. but this value falls to the adult range by the end of the 2nd week of life. The reticulocyte count is of value as an indication of a shorter-than-normal erythrocyte survival, which is based on the deduction that the total red blood cell (RBC) mass in a steady state is equal to the number of new RBCs produced, multiplied by the 120-day life span of individual cells. When the RBC mass falls, it is the result of decreased RBC production or a shortened life span. Normal erythropoiesis corrects for a shorter life span by increasing the production rate, which the reticulocyte count measures. An elevated reticulocyte count accompanies a shortened RBC survival. Reticulocytosis indicates that the body is trying to maintain homeostasis. Calculating and Expressing Traditional Reticulocyte Values Traditionally, the reticulocyte count has been expressed as a percentage of the total number of circulating erythrocytes (e.g., 1%). However, this relative value may be erroneous because fluctuation in the percentage may be caused by a change in the total number of circulating erythrocytes rather than a true change in the number of circulating reticulocytes. To account for variations caused by erythrocyte quantity, expression of reticulocytes in absolute rather than proportional terms is becoming the preferred method of reporting. The correction for anemia is helpful for clinical interpretation, and several different methods are used. The Clinical Laboratory Standards Institute (CLSI) proposes that the correction for anemia, the corrected reticulocyte count, be made mathematically by correcting the observed reticulocyte count to a normal packed RBC volume (hematocrit). Corrected Reticulocyte Count Example: If an adult male has a hematocrit of 30% (0.30 L/L) and a reticulocyte count of 3%, the corrected reticulocyte count would be The normal value based on correction for anemia is the same as the previously stated normal reticulocyte values of 0.5% to 1.5%. Reticulocyte Production Index A simple percentage calculation of reticulocytes does not account for the fact that prematurely released reticulocytes require from 0.5 to 1.5 days longer in the circulating blood to mature and lose their netlike reticulum. The reticulocyte count, even if corrected, will be elevated out of proportion to the actual increase in erythrocyte production because of the accumulation of these younger reticulocytes in the circulating blood. An older method to correct for immature reticulocytes is the reticulocyte production index (RPI). This method was used before other measurements were available using automated instruments. The RPI measures erythropoietic activity when stress reticulocytes are present. The rationale for obtaining this value is that the life span of the circulating stress reticulocytes is 2 days instead of the normal 1 day. To compensate for the increased maturation time and consequent retention of residual RNA of the prematurely released reticulocytes, the corrected reticulocyte count is divided by a correction factor derived from the maturation timetable (Table 10.3). The CLSI recommends replacing the RPI with other reticulocyte parameters. TABLE 10.3 Maturation Time Correction Factor Hematocrit (%) Maturation Time (Days) 45 1.0 35 1.5 25 2.0  15 2.5 Calculation of the Reticulocyte Production Index If the corrected reticulocyte count is 2.0% and the patient’s hematocrit is 0.30 L/L, the RPI is Absolute Reticulocyte Count This is a classic measurement of the bone marrow erythropoietic response, reticulocyte production, in conditions of anemia. The absolute reticulocyte count is the actual number of reticulocytes in 1 liter (L) or 1 microliter (μL) of blood. The normal reference range is 25 to 75 (20 to 115) × 109/L. If the absolute reticulocyte value is less than 100 × 109/L, the patient has an inappropriately low erythropoietic response to his or her anemia. Although automated analyzers can report the absolute reticulocyte, the manual formula for calculating the absolute reticulocyte count is *The calculated result has to be converted from 1012/L to 109/L. Example: A 35-year-old female diagnosed with anemia has an erythrocyte (RBC) count of 2.7 × 1012/L, a 29% hematocrit, and a reticulocyte count of 2.0%. Her absolute reticulocyte count is In this case, the patient’s bone marrow is not adequately compensating for her anemia. New Reticulocyte Assays More recently, automated methods based on flow cytometry have become widely utilized. The automated methods count a larger number of cells and exhibit a greater degree of reproducibility. The normal reference range by these methods extends to a lower reticulocyte value and may reflect detection of white cell fragments, which sacrifices the accuracy of very low reticulocyte counts Automated instruments provide relative and absolute reticulocyte and assessment of reticulocyte maturity level as a semiquantitative measurement of RNA concentration in maturing erythrocytes Because younger reticulocytes contain more RNA than do more mature RBCs, the term immature reticulocyte fraction (IRF) is used for this purpose. The IRF or reticulocyte maturity index are new parameters that enable a comparison between immature reticulocytes, which contain the most RNA, and mature reticulocytes. The IRF is useful in assessing bone marrow response in cases of recovery from chemotherapy or posttransplantation engraftment. Evaluation of the bone marrow erythropoietic response is helpful in cases of treated anemias such as iron deficiency or B12 administration to patients with pernicious anemia. In these cases, the IRF will increase before a reticulocyte count or an increase in hemoglobin, hematocrit, or red blood cell count. Another component generated by automated instruments is the reticulocyte hemoglobin assay. The measurement of reticulocyte hemoglobin is a measurement of the hemoglobin content of reticulocytes that reflects the availability of functional iron for the red cell and the incorporation of iron in the synthesis of the hemoglobin molecule over the last several days. The reticulocyte hemoglobin indicates response or the lack of response to iron therapy. Another reticulocyte measurement is reticulated hemoglobin content (CHr). CHr hemoglobin content of retics analogous to MCH. The red blood cell mean reticulocyte cell hemoglobin concentration is equivalent to the MCHC. Assessment of Bone Marrow Response Normal bone marrow activity produces an RPI index of 1. In hemolytic anemias, in which there is increased destruction of erythrocytes in the peripheral blood and a functionally normal marrow, this index may be three to seven times higher than normal. In cases of bone marrow damage, erythropoietin suppression, or a deficiency of vitamin B12, folic acid, or iron (hypoproliferative states), the index is 2 or less. NOTE: This is a good time to complete Review Questions related to the preceding content. QUANTITATIVE ASSESSMENT OF LEUKOCYTES Total Leukocyte Count The calculation of a manual total leukocyte count is Total WBC count = average total WBCs × dilutional correction factor × volume correction factor 1. The number of total leukocytes is the average of the two sides of the hemacytometer. 2. The dilutional factor is 20 based on the dilution of 1:20 of the blood specimen. 3. The volume correction factor is 2.5. This represents the volume desired (1.0 μL) divided by the volume counted (0.4 μL). Example: If the average total of leukocytes counted was 180, the total leukocyte count would be: A simplification of this formula is to use a factor of 50 × the dilution factor of 20 × the volume correction factor of 2.5. Example: 180 × 50 = 9.0 × 109/L. Sources of error include contaminated diluting fluid, incorrect diluting or loading of the hemacytometer, and an uneven distribution of leukocytes in the counting chamber. Clean, dry pipettes and prompt counting of cells are important to the accuracy of the count. The normal WBC count for adults varies from 4.5 to 11.0 × 109/L. An increase in the WBC (leukocyte) count above the normal upper limit is termed leukocytosis. A decrease below the normal lower limit is termed leukopenia. Leukopenia may occur with certain viral infections, with typhoid fever and malaria, after radiation therapy, and in classic pernicious anemia. A sudden drop (within a few days) in the total WBC count from normal to a state of leukopenia can be observed after chemotherapy treatments. The total leukocyte count (see procedure in Chapter 32, Laboratory Manual) can be elevated, leukocytosis, above 10 × 109/L in conditions such as pregnancy or strenuous exercise. A diagnosis of acute inflammation is generally based on a total leukocyte count greater than 10.5 × 109/L in combination with other factors. The total count may be depressed, leukocytopenia, because of overwhelming bacterial infection (sepsis) or immunosuppressive agents. Leukocytosis may occur in many acute infections, especially bacterial infections, in severe malaria, after hemorrhage, during pregnancy, postoperatively, in some forms of anemia, in some carcinomas, and in leukemia. Inflammation almost always follows acute tissue damage. Diagnostic categories of acute inflammation can include bacterial causes and nonbacterial causes such as trauma, chronic inflammation, and viral disease. Among the many laboratory tests that have been advocated for the diagnosis of inflammation, the total leukocyte count, the percentage of band and segmented neutrophils determined by a differential leukocyte count (see peripheral blood film evaluation below), the absolute neutrophil cell count, and the erythrocyte sedimentation rate (ESR) are the most common. Absolute Cell Counts The absolute number of segmented neutrophils and bands is considered to be a less specific index of inflammation than other tests because the total leukocyte count drops in many patients with overwhelming infection. This condition results from the movement of circulating granulocytes into the tissue sites of infection. An absolute cell count may be valuable in other cases of inflammation. An example of the method of calculating an absolute cell count is presented in Box 10.1. In other situations, an absolute lymphocyte count is helpful. BOX 10.1 Absolute Cell Counts Absolute count* = absolute cell value = total leukocyte count × percentage of cell type PATIENT DATA Total leukocyte count: 15.0 × 109/L Differential blood smear results: bands 12%, segmented neutrophils 80%, lymphocytes 8% SAMPLE CALCULATION Absolute segmented neutrophil value = total leukocyte count × % of segmented neutrophils Absolute value = 15.0 × 109/L × 0.80 = 12.0 × 109/L segmented neutrophils *This formula can be used to determine the absolute value of any cell appearing on a leukocyte differential blood smear. Normal adult absolute values include segmented neutrophils 1.4 to 6.5 × 109/L, bands 0 to 0.7 × 109/L, lymphocytes 1.2 to 3.4 × 109/L. Assessment of Eosinophils and Basophils Examination of a peripheral blood smear normally demonstrates an average of approximately 4% eosinophils. An increase in eosinophils, eosinophilia, can be observed in active allergies and some parasitic infections. Because this method of estimation is only semiquantitative, an absolute eosinophil count, either by manual chamber counting or by the use of automated equipment, is preferred. This procedure is required only if an extreme increase in eosinophils is demonstrated on a peripheral blood smear or if clinical symptoms suggest an increase. OTHER LEUKOCYTE-RELATED ASSESSMENTS Neutrophilic Hypersegmentation Index Mature segmented neutrophils have two to five nuclear lobes (segments). Counting the number of lobes can be performed to determine the neutrophilic hypersegmentation index (NHI). A right shift or increase in the number of lobes to five or more occurs in various conditions, for example, sepsis, chronic nephritis. The NHI is clinically useful in vitamin B12 deficiency (pernicious anemia) and folic acid diagnosis. Three methods exist for calculating the NHI: 1. Lobe average. This is determined by counting the number of lobes in a number of neutrophils, for example, 200, and dividing by the total number of neutrophils for the average number of lobes. The reference value is 2.5 to 3.3. 2. Percentage of neutrophils with five or more lobes. Count the number of lobes in randomly selected segmented neutrophils, for example, 200. Add up the total number of lobes for each segmented neutrophil counted, and divide by the total number of cells counted. The reference range is greater than 3%. 3. Hypersegmentation index. To calculate this index, use a minimum of 200 segmented cells. Values greater than 16.9 are considered to indicate hypersegmentation. This method is considered to be the most sensitive method. The basophil is the least numerous of the granulocytes. Normally, differential smears of normal blood have only 1% basophils, if any. An increase in basophils, basophilia, is very significant and is seen in conditions such as chronic myelogenous leukemia and polycythemia vera. Neutrophilic Function A number of diseases are associated with leukocyte dysfunctions related to locomotion, chemotaxis, adhesion, or the ability of cells to destroy infectious organisms. In vitro assays of the rate of cell movement and the directional orientation of the movement as well as the ability of granulocytes to destroy organisms have been in existence for more than 20 years. A defect in cell adhesiveness, for example, leukocyte adhesion defect (LAD), can lead to decreased cell locomotion.  A test that assesses the killing ability of granulocytes is the nitroblue tetrazolium (NBT) test. In the routine clinical laboratory, this procedure is infrequently performed. Functional abnormalities expressed by patients with congenital neutropenia include defective migration, bacterial killing, or increased apoptosis. Leukocyte Alkaline Phosphatase Test Performance of this procedure is discussed in detail in Chapter 32. The value of this cytochemical stain is in differentiating malignant disorders from leukemoid reactions. Erythrocyte Sedimentation Rate (ESR) Except for some refinements, the eryhrocyte sedimentation rate (ESR) procedure continues to be an established parameter of inflammation in the modern clinical laboratory. The Westergren method has been selected by the CLSI as the standard method of choice. The ESR, or sed rate, is a nonspecific indicator of disease. Although this procedure is nonspecific, it is one of the most commonly performed laboratory tests. Very few tests have as long a history as does the ESR. A Swedish physician, Fahraeus, is credited with the discovery of this test in 1915. However, the sedimentation of blood was one of the principles on which ancient Greek medicine was based. The Greek philosophy of the four humors (fluids) in the human body was established in the 5th century BC and further developed by Aristotle. This belief proposed that these fluids formed the body. On the basis of this philosophy, each person had a predisposition for a particular disease depending on the predominance of one of the four fluids: blood, phlegm, yellow bile, or black bile. In 1836, Nasse recognized that a property of plasma, later identified as increased proteins, produced an increased sinking speed of erythrocytes in whole blood. The work of Nasse went unnoticed for nearly a century because medicine was undergoing a radical reform, moving away from the humoral philosophy of the Greeks toward the cellular pathology theories of Virchow. With the reestablishment by Fahraeus of the significance of the empirical basis of Greek medicine, Westergren began working concurrently on refining the technique. The reference value of this test varies depending on age. In persons younger than 50 years of age, the average reference values are up to 10 mm/hour in males and 13 mm/hour in females. For persons older than 50 years of age, average reference values are up to 13 mm/hour in males and up to 20 mm/hour in females. Erythrocytes with abnormal or irregular shapes, such as sickle cells or spherocytes, hinder rouleaux formation and lower the ESR. The removal of fibrinogen by defibrination also produces a decreased ESR. An increased ESR value can be observed in various abnormal blood conditions: rouleaux, increased fibrinogen levels, a relative increase of plasma globulins caused by the loss of plasma albumin, and an absolute increase of plasma globulins. Clinical conditions associated with increased ESR values include anemia, infections, inflammation, tissue necrosis (such as myocardial infarction), pregnancy, and some types of hemolytic anemia. NOTE: This is a good time to complete Review Questions related to the preceding content. QUANTITATIVE ASSESSMENT OF PLATELETS Another formed element of the blood is the platelet, or thrombocyte. Platelets are produced in the bone marrow by cells called megakaryocytes, which are large and multinucleated. Platelets do not have a nucleus and are not actually cells; they are portions of cytoplasm pinched off from megakaryocytes and released into the bloodstream. Mature platelets are small, colorless bodies 1.5 to 4 μm in diameter. Platelets are generally round or ovoid, although they may have projections. Platelets have a colorless to pale-blue background substance containing centrally located, purplish red granules. The circulating platelet count can be accurately determined in an anticoagulated blood specimen using an electronic particle counter (see Chapter 30). Most platelet counts are performed using automated instruments. The quantitative platelet count is correlated with a semiquantitative estimate from a stained peripheral blood smear. If the instrument count and the blood smear do not match, a manual platelet count is performed. This situation rarely happens when the platelet count is very low and the patient has a moderate number of schistocytes. Platelet Count The circulating platelet count can be accurately determined in a blood sample using an electronic particle counter. The traditional manual platelet count using a pre- measured reservoir mixes and the whole blood is diluted 1:100 with 1% oxalate buffer solution. The diluent lyses all RBCs and disaggregates the platelets. With phase contrast microscopy, the platelets appear as dark round or oval bodies because of their higher refractive index. The chamber counting method of platelet enumeration allows direct visualization of the particles being counted. It does not have a high degree of precision or a high degree of accuracy. It is prone to technique variation. If blood is diluted 1:100 and a manual platelet count is performed, the number of platelets is counted on each side of a hemacytometer’s red blood cell counting area. The average of the two counts is determined and expressed in a numerical value × 109/L. The normal reference range is 150 to 450 × 109/L. Example: If 200 platelets are counted on one side of the appropriate counting area and 210 platelets are counted on the other side, what is the total platelet count? 200 + 210 = 410/2 = 205 × 109/L. Calculation: When platelets are counted in the large center square (1 mm2) using high power (40×), the following formula is used to calculate the total platelet count.  Number of platelets per microliter is as follows: Platelets/μL = total average number of platelets in 5 squares × dilution correction factor × volume correction factor Example: If the average total of platelets counted in five squares is 20, the platelet count is 20 × 200 × 50 = 200 × 109/L The average adult circulating platelet concentration is the total number of cells counted must be corrected for the initial dilution of blood and the volume of diluted blood used. The standard dilution of blood for platelet counts is 1:100; therefore the dilution factor is 100. The volume of diluted blood used is based on the area and depth of the counting area. 109/L. Various patient conditions can produced an increased or decreased concentration of platelets. A platelet disorder, immune thrombocytopenia can produce a decrease in the total platelet count. A decrease can also be seen in anemias such as aplastic anemia and megaloblastic anemia. Other causes of decreases can be acute leukemia and myelodysplastic syndromes. Increases in the total concentration of platelets in the circulation can result from splenectomy. Other pathologic causes of thrombocytosis are myeloproliferative neoplasms, acute hemorrhage, and iron deficiency anemia. Sources of Error: A variety of technical errors can produce incorrect results. These include the age of the specimen, clumping of platelets, debris in the diluting fluid, platelet adherence to glass, and incorrect dilution of the specimen. An even distribution of platelets through the counting is critical. Clumping results from inadequate mixing or poor technique. If clumps are seen, the sample must be rediluted and recounted. In cases of thrombocytopenia, the dilution may have to be increased to 1:100 or 1:20. If a small clot inadvertently forms in an EDTA-anticoagulated blood specimen, it can have an adverse effect on the total platelet count and platelet count estimated. Small blood clots are composed of platelets and fibrin strands. If a small clot exists in a blood specimen, the platelet count will be falsely decreased. PERIPHERAL BLOOD FILM EVALUATION Proper blood specimen collection of either venous or capillary blood, blood film preparation, and staining of the blood film are discussed in Chapter 2. The starting point for the evaluation of a blood film is a well-prepared and a well stained blood smear. Direct visual observation should show a pink to purple color. Upon microscopic examination, the blood film should show RBCs as slightly orange to dark pink structures; WBCs should exhibit nuclei with a dark blue to purple color. Granulocytic cells have various colored granules in the cytoplasm. Neutrophilic granules should appear as a violet color. The two populations of eosinophilic granules are solid, dark orange and crystalline, light orange. Basophilic granules are dark blue in color. Microscopic Examination The care and use of the microscope is discussed in Appendix G. Once the proper focusing has been achieved with the low power (10×) lens, the objective can be moved to high power (40×) and to oil immersion (100×). Each level of magnification can reveal different aspects of the blood smear (see Table 10.4). TABLE 10.4 Observations at Various Microscopic Magnifications Description Low Power (10×) This is the usual beginning point for the examination of a stained blood film. At low power magnification, the overall blood smear quality, color, and distribution of cells can be assessed. If the feather edge or slides of the blood smear have crowding of cells compared to the middle areas of the slide, the slide is unacceptable. The sections of the stained blood film where the RBCs are barely touching each other can be inspected for the presence of rouleaux or agglutination can be indicative of a variety of disorders (see Table 10.5). Large immature or abnormal cells or disintegrating lymphocytes, smudge cells, may also be detected on low power. Clumping of platelets or platelet satellitism may also be observed.  TABLE 10.5 Red Blood Cell Distribution Disorders Terminology Description rouleaux Overlapping red blood cells, often referred to as a “stack of coins” caused by abnormal or increased plasma proteins. May be an artifact, if anticoagulated blood specimen is allowed to stand for hours or if the thick part of blood smear is examined. agglutination Clumps of red blood cells looking like bunches of grapes. This condition can falsely decreased the automated, calculated hematocrit and falsely increase the MCH and MCHC. High Power 40× After moving from the low power 10× objective to high power (40×), select the correct area of the blood film, the area where the RBCs are barely touching each other, the total leukocyte (WBC) concentration can be estimated. Oil Immersion Lens (1,000×) After switching to 100× (oil) and locating an area where the RBCs are just touching or overlapping, a leukocyte differential can be performed using an organized tracking system. This process involves counting 100 leukocytes and differentiating them into different categories as well as observing red blood cell morphology and the distribution of platelets (see Figs. 10.1 and 10.2). FIGURE 10.1 Normal peripheral blood smear. The mature erythrocytes are of normal size and show central pallor. A neutrophil and monocyte are also present. (Reprinted from Rubin E, Farber JL. Pathology, 3rd ed, Philadelphia, PA: Lippincott Williams & Wilkins, 1999, with permission.) FIGURE 10.2 Peripheral blood smear showing morphologically normal RBCs, platelets, and a small lymphocyte. (Reprinted from Topol EJ, et al. Textbook of Cardiovascular Medicine, 3rd ed, Philadelphia, PA: Lippincott Williams & Wilkins, 2006, with permission.) SEMIQUANTITATIVE GRADING OF ERYTHROCYTE MORPHOLOGY Direct observation of a peripheral blood smear for abnormalities in erythrocytic morphology or immature erythrocytes can yield additional information. In addition to the identification of erythrocyte abnormalities (discussed in Chapter 7), erythrocyte morphology may be reported semiquantitatively to reflect the severity of the abnormalities. Variation in normal RBC size is anisocytosis; variation in normal RBC shape is poikilocytosis. Erythrocyte changes are commonly reported using either descriptive terms, such as moderate or marked, or grades on a numerical scale, such as 1+, 2+, 3+, or 4+. The characteristics of such a grading scale may vary from one laboratory to another but will generally conform to the scale as presented in Table 10.6. TABLE 10.6 Grading of Erythrocyte Morphology Numerical Scale Description 0 Normal appearance or slight variation in erythrocytes. 1+ Only a small population of erythrocytes displays a particular abnormality; the terms slightly increased or few would be comparable. 2+ More than occasional numbers of abnormal erythrocytes can be seen in a microscopic field; an equivalent descriptive term is moderately increased. 3+ Severe increase in abnormal erythrocytes in each microscopic field; an equivalent descriptive term is many. 4+ The most severe state of erythrocytic abnormality, with the abnormality prevalent throughout each microscopic field; comparable terms are marked or marked increase. SEMIQUANTITATIVE ASSESSMENT OF LEUKOCYTES A semiquantitative estimate of WBCs to quantitative measurements of WBCs is a good quality control step. This may detect specimen errors. In some cases, estimates are used only as needed to confirm instrument values particularly low counts. A summary of the process of performing a white blood cells estimate is presented in Box 10.2. BOX 10.2 Total White Blood Cell Estimate 1. Select an appropriate area of the stained blood smear at 40× magnification (RBCs just touching). 2. Count the number of WBCs in each of ten fields (minimum). 3. Calculate the estimated total concentration of WBCs per μL by multiplying the average number of WBC/field × 2,000. 4. Compare this estimate with the quantitatively determined automated or manual WBC count Example: If an average of 8 WBCs are observed per field with a 40× objective, the total concentration is calculated by multiplying 8 × 2,000. The WBC estimate is 16,000/μL or mm3 or 16.0 × 109/L. Source: Modified from Terrell JC. Laboatory ealuation of leukocytes. In: Stiene-Martin EA, Lotspeich- Steininger CA, Koepke JA (eds.). Clinical Hematology: Principles, Procedures, Correlations, 2nd ed, Philadelphia, PA: Lippincott-Raven Publishers, 1998:337. SEMIQUANTITATIVE ASSESSMENT OF PLATELETS A platelet count is a fundamental component in the evaluation of a patient. Examination of the peripheral blood smear for an estimate of the number of platelets in circulation and morphology is critical because many clinical clues may be obtained from an evaluation of platelet quantity and morphology. Examination of a stained blood film provides a rapid estimate of platelet numbers. Although the estimation of platelets from a blood smear does not replace an actual quantitative measurement, it should be done as a cross-check of the quantitative measurement. Normally, 8 to 20 platelets are present in an oil immersion field in a properly prepared smear (where the RBCs barely touch each other). After examining at least 10 different fields, the average number of platelets can be multiplied by a factor of 20,000 to arrive at an approximate total circulating platelet concentration. Example: If an average of 14 platelets are observed per field in ten different fields with a 100 × objective, the total concentration is calculated by multiplying 14 × 20,000. The platelet estimate is 280,000/μL or mm3 or 280.0 × 109/L. Note: If a significant increase in the number of red blood cells, erythrocytosis, or an anemia exists, this formula should be used: LEUKOCYTE DIFFERENTIAL COUNT Principle A stained smear is examined to determine the percentage of each type of leukocyte present and assess the erythrocyte and platelet morphology. Increases in any of the normal leukocyte types and the presence of immature leukocytes or erythrocytes in peripheral blood are important diagnostically in a wide variety of inflammatory disorders and leukemia. Erythrocyte abnormalities are clinically important in various anemias. Platelet size irregularities are suggestive of particular thrombocyte disorders. Specimen Peripheral blood, bone marrow, or body fluid sediments, such as spinal fluid, are appropriate specimens. Whole blood smears may be made from EDTA- anticoagulated blood or prepared from free-flowing capillary blood. Smears should be made within 1 hour of blood collection from EDTA specimens stored at room temperature to avoid distortion of cell morphology. Unstained smears can be stored for indefinite periods, but stained smears gradually fade. Reagents, Supplies, and Equipment 1. A manual cell counter designed for differential counts 2. Microscope, immersion oil, and lens paper Quality Control Training and experience in examining immature and abnormal cell morphology are essential. A set of reference slides with established parameters should be established to assess the competence of an individual to perform differential and morphological identification of leukocytes and erythrocytes. Participation in a quality assurance program continues to document the expertise of the hematologist in microscopy. Questionable or abnormal smears should be referred to a supervisor for verification. Procedure 1. Begin the slide examination with a correctly prepared and stained smear (see Chapter 2 for specimen preparation). 2. Focus the microscope on the 10× objective (low power). Scan the smear to check  for cell distribution, clumping, and abnormal cells. Add a drop of immersion oil and switch to the 100× (oil immersion) objective. Begin the count by determining a suitable area (Fig. 10.1). Extend the examination from the area where approximately half of the erythrocytes are barely overlapping to an area where the erythrocytes touch each other. It is important to examine cellular morphology and to count leukocytes in areas that are neither too thick nor too thin. In areas that are too thick, cellular details such as nuclear chromatin patterns are difficult to examine. In areas that are too thin, distortion of cells makes it risky to identify a cell type. 3. A total of at least 100 leukocytes should be counted. Express the results as a percentage of total leukocytes counted. Count the leukocytes using a tracking pattern (see Fig. 32.2). Each cell identified should be immediately tallied as a neutrophil (band), neutrophil (segmented), or polymorphonuclear neutrophil (PMN); lymphocyte; monocyte; eosinophil; or basophil. A brief leukocyte morphology reference is included (Table 10.7). Refer to chapters 7,8 and 9 for a complete discussion on leukocyte and erythrocyte cellular morphology. TABLE 10.7 A Comparison of Normal Leukocytes in Peripheral Blood Description 4. Abnormalities of leukocytes, erythrocytes, and platelets should be noted. Nucleated erythrocytes are not included in the total count but are noted per 100 white blood cells (see correction formula). Reporting Results Reference values, particularly the band neutrophil percentage, may vary. Values for children differ from adult reference values. See inside back cover for a full discussion of reference values. Procedure Notes The blood smear preparation techniques described in Chapter 2 are commonly used in the laboratory for the preparation of blood smears. In circumstances where the WBC count is extremely low, the preparation of a buffy coat (see procedure in Chapter 32) increases the accuracy of the leukocyte differential count. The knowledge and ability of the cell morphologist are critical to high-quality results. The morphology of erythrocytes, leukocytes, and platelets need to be observed for deviations from normal appearance. Changes in cellular appear can be indicative of a hematologic disorder. In some cases, changes in cellular appearance may be due to technical errors such as failure to prepare the blood smear collected in EDTA within a few hours of blood collection or underfilling an evacuated collection tube, which can result in excess anticoagulant and induced changes. Changes due to technical errors include alteration of the cellular nucleus, degranulation, or cytoplasmic vacuoles, Some abnormalities detected on a peripheral blood smear may have an effect on total cell counts, and corrective procedures may be needed (Box 10.3). BOX 10.3 Technical Issues: Total Cell Counts and Corrective Action Cell Type False Impact on Resolution Total Cell Count >5 Elevated WBC Calculate WBC correction, if not Nucleated automatically corrected by blood RBCs/100 cell counting instrument. WBCs Agglutination Decreased RBC Warm blood specimen to 37°C for of RBCs count 15 minutes and then retest. Rouleaux None No correction available. Platelet Decreased platelet If collected in EDTA anticoagulant, clumps or count redraw blood specimen in citrate platelet anticoagulant and multiply the satellitism platelet count by a factor of 1.1 to correct for dilutional effect of liquid citrate anticoagulant. Alternate strategy: Obtain a capillary blood specimen and prepare blood smear at the patient’s  point of care. A minimum of 300 leukocytes must be within the acceptable working area, when the total leukocyte count is no less than 4 × 109/L. The neutrophils, monocytes, and lymphocytes should appear evenly distributed in the usable fields of the film. Less than 2% of the leukocytes should be disrupted or nonidentifiable forms except in certain forms associated with pathological states. If a disrupted cell is clearly identifiable, include it in the differential count. Classify nonidentifiable disrupted cells (smudges or baskets) as “other,” and note them on the report if more than a few are observed. The presence of nucleated red blood cells in the peripheral blood circulation demonstrates disruption of normal bone marrow release of mature cells into the circulation. If more than 5 nucleated erythrocytes are observed in a 100 cell differential count, the total white blood cell count must be corrected (see Box 10.4). Some automated blood cell counters recognize and correct for the presence of nucleated red blood cells. BOX 10.4 Formula for Leukocyte Count Correction* * If more than 5 nucleated red blood cells/100 white blood cells at 1,000× (100×) magnification. Example: Various disorders associated with an increases in the distribution of normal types of leukocytes (see Box 10.5). BOX 10.5  Disorders Associated with Increases in Distribution of normal types of leukocytes NEUTROPHILS 1. Bacterial infections 2. Inflammation 3. Stress 4. Chronic leukemia LYMPHOCYTES 1. Viral infections 2. Whooping cough 3. Chronic leukemia MONOCYTES 1. Tuberculosis 2. Rheumatoid arthritis 3. Fever of unknown origin EOSINOPHILS 1. Active allergies 2. Invasive parasites BASOPHILS 1. Ulcerative colitis 2. Hyperlipidemia Patients with stress conditions can demonstrate an increase in the number of band forms in the presence of a normal total leukocyte count. The normal average for band neutrophils is considered to be 3% in adults; newborn infants have a somewhat higher normal average. A neutrophilic band count greater than 11% is considered to be consistent with an inflammatory condition. The normal average for segmented neutrophils is 56% and approximately 4% for monocytes. Shift to the Left When the percentage of band forms and other immature neutrophils such as metamyelocytes and myelocytes increases, the condition is sometimes referred to as a shift to the left. The terminology, shift to the left, dates back to the 1920s and lives on today to describe increased numbers of immature neutrophilic cells as an indicator of infection. Some authorities advocate doing away with the identification of band forms on the leukocyte differential procedure because of individual variability in cell identification and limited usefulness. The Clinical Laboratory Standards Institute recommends that bands and neutrophils be counted together and placed in a single category rather than in separate categories because of the individual variability in the differentiation of band form of neutrophils. NOTE: This is a good time to complete the end of chapter Review Questions related to the preceding content. CHAPTER HIGHLIGHTS The Complete Blood Count (CBC) A CBC consists of specific measurements of hemoglobin, hematocrit, red and white blood cell counts, platelet count, leukocyte differential and evaluation of a peripheral blood smear as basic information. Quantitative measurements of erythrocytes, leukocytes, and platelets are a standard part of the report generated by automated instrumentation. In most cases, RBC or WBC counts are only performed manually when there are extremely low total leukocyte counts from whole blood or body fluid specimens. Manual cell counting may be used for counting cells from body fluids, such as cerebrospinal fluid or synovial fluid. The RBC indices of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) are now a standard part of a routine automated CBC. Through the use of automated hematology instrumentation, additional measurements have been added to a report, including reticulocyte information, red cell distribution width (RDW), and blood cell histograms. In manual cell counting, blood specimens are diluted to an exact ratio with specific diluents and the cells are counted in a hemacytometer, an accurately ruled chamber ruled off in areas of square millimeters. Quantitative Assessment of Erythrocytes The determination of hemoglobin (Hb) is by the hemiglobincyanide (cyanmethemoglobin) method. The hematocrit (Hct), or packed cell volume, is a macroscopic observation of volume of the packed RBCs in a sample of whole blood. A manual RBC count involves calculating the average total number of cells in 5 squares of a hemacytometer and multiplying by the dilution correction factor and the volume correction factor. The erythrocyte indices are used to mathematically define cell size and the concentration of hemoglobin within the cell. RBC indices are MCV to express the average volume of an erythrocyte, MCH to expresses the average weight (content) of hemoglobin in an average erythrocyte, and MCHC to express the average concentration of hemoglobin per unit volume of erythrocytes or the ratio of the weight of hemoglobin to the volume of erythrocytes. As an erythrocyte develops, the nucleus becomes more and more condensed and is eventually lost. After the loss of the nucleus, an immature erythrocyte remains in the bone marrow for 2 to 3 days before entering the circulating blood. During this period in the bone marrow and during the first day in the circulation, this immature erythrocyte is referred to as a reticulocyte. The loss of ribosomes and mitochondria, along with full hemoglobinization of the cell, marks the transition from the reticulocyte stage to full maturation of the erythrocyte. Under normal conditions, the quantity of reticulocytes in the bone marrow is equal to that of the reticulocytes in the circulating blood. Because the normal life span or survival time is 120 days, 1/120th of the total number of erythrocytes is lost each day, and an equal number of reticulocytes is released into the circulation. If, under the stimulus of erythropoietin, increased numbers of young reticulocytes are prematurely released from the bone marrow because of such conditions as acute bleeding, these reticulocytes are referred to as stress or shift reticulocytes. Peripheral smears of normal blood stained with Wright stain may demonstrate a slight blue tint in some erythrocytes referred to as polychromatophilia or polychromasia. A supravital stain, such as new methylene blue, precipitates the ribosomal RNA in immature red blood cells to form a deep-blue, meshlike network. The reticulocyte count is expressed as a percentage of total erythrocytes. The normal range is 0.5% to 2.0% in adults. In newborn infants, the range is 2.5% to 6.0%, but this value falls to the adult range by the end of the 2nd week of life. To account for variations caused by erythrocyte quantity, expression of reticulocytes in absolute rather than proportional terms is becoming the preferred method of reporting. Because younger reticulocytes contain more RNA than do more mature RBCs, the term immature reticulocyte fraction (IRF) is used for this purpose. It is helpful in the evaluation of the bone marrow erythropoietic response to treatment. Another component generated by automated instruments is the reticulocyte hemoglobin assay. This is a measurement of the hemoglobin content of reticulocytes that reflects the availability of functional iron for the red cell and the incorporation of iron in the synthesis of the hemoglobin molecule over the last several days. Another reticulocyte measurement is reticulated hemoglobin content (CHr). CHr hemoglobin content of retics is analogous to MCH. The red blood cell mean reticulocyte cell hemoglobin concentration is equivalent to the MCHC. Normal bone marrow activity produces an equivalent number of reticulocytes. In hemolytic anemias, where there is increased destruction of erythrocytes in the peripheral blood and a functionally normal marrow, erythropoiesis may be extremely increased. In cases of bone marrow damage; erythropoietin suppression; or a deficiency of vitamin B12, folic acid, or iron, erythropoiesis is  below the RBC replacement level. Quantitative Assessment of Leukocytes The total leukocyte manual count involves multiplying the average total WBCs × dilutional correction factor × volume correction factor. Inflammation almost always follows acute tissue damage. Acute inflammation can include bacterial causes and nonbacterial causes such as trauma, chronic inflammation, and viral disease. Among the many laboratory tests that have been advocated for the diagnosis of inflammation, the total leukocyte count, the percentage of band and segmented neutrophils determined by a differential leukocyte count, the absolute neutrophil cell count, and the erythrocyte sedimentation rate (ESR) are the most common. Leukocytosis, an elevation of the total granulocyte count above the normal reference range, can be observed in conditions such as pregnancy or strenuous exercise. Leukocytopenia can result from overwhelming bacterial infection (sepsis) or immunosuppressive agents. Absolute cell counts can be calculated for any of the individual blood cell types. The absolute number of segmented neutrophils and bands is considered to be a less specific index of inflammation than other tests because the total leukocyte count drops in many patients with overwhelming infection. Eosinophilia can be observed in active allergies and some parasitic infections. Basophilia is seen in conditions such as chronic myelogenous leukemia and polycythemia vera. Mature segmented neutrophils have two to five nuclear lobes. Counting the number of lobes can be performed to determine the neutrophilic hypersegmentation index (NHI). A right shift or increase in the number of lobes to five or more occurs in various conditions, for example, sepsis, chronic nephritis. The NHI is clinically useful in vitamin B12 deficiency (pernicious anemia) and folic acid diagnosis. A number of diseases are associated with leukocyte dysfunctions related to locomotion, chemotaxis, adhesion, or the ability of cells to destroy infectious organisms. A defect in cell adhesiveness, for example, leukocyte adhesion defect (LAD), can lead to decreased cell locomotion. Functional abnormalities expressed by patients with congenital neutropenia include defective migration, bacterial killing, or increased apoptosis. Leukocyte alkaline phosphatase (LAP) test is a cytochemical stain used to differentiated malignant disorders from leukemoid reactions. The erythrocyte sedimentation rate (ESR), or sed rate, is a nonspecific indicator of disease with increased sedimentation of erythrocytes in acute and chronic  inflammation and malignancies. The ESR procedure continues to be an established parameter of inflammation in the modern clinical laboratory. The Westergren method has been selected by the CLSI as the standard method of choice. Quantitative Assessment of Platelets Another formed element of the blood is the platelet, or thrombocyte. Most platelet counts are performed using automated instruments. The quantitative platelet count is correlated with a semiquantitative estimate from a stained peripheral blood smear. Peripheral Blood Film Evaluation The starting point for the evaluation of a blood film is a well stained blood smear. Upon microscopic examination, the blood film should show RBCs as slightly orange to dark pink structures; WBCs should exhibit nuclei with a dark blue to purple color. Direct observation of a peripheral blood smear for abnormalities in erythrocytic morphology or immature erythrocytes can yield additional information. Variation in normal RBC size is anisocytosis; variation in normal RBC shape is poikilocytosis. A comparison of a semiquantitative estimates of WBCs to quantitative measurements of WBCs is a good quality control step. This comparison can detect specimen errors, slide labeling errors, or errors in the quantitative measurement of these cells. In some cases, estimates are used only as needed to confirm instrument values. Checking for discrepancies can potentially detect specimen errors. Normally, there are 8 to 20 platelets per 100× (oil) immersion field in a properly prepared smear from an anticoagulated blood specimen. At least 10 different fields should be carefully examined for a semiquantitative platelet estimation. Granulocytic cells have various colored granules in the cytoplasm. Neutrophilic granules should appear as a violet color. The two populations of eosinophilic granules are solid, dark orange and crystalline, light orange. Basophilic granules are dark blue in color. When the percentage of band forms and other immature neutrophils such as metamyelocytes and myelocytes increases, the condition is sometimes referred to as a shift to the left. The Clinical Laboratory Standards Institute recommends that bands and neutrophils be counted together and placed in a single category rather than in separate categories because of the individual variability in the differentiation of band form of neutrophils. Patients with stress conditions can demonstrate an increase in the number of band forms in the presence of a normal total leukocyte count. The normal average for band neutrophils is considered to be 3% in adults; newborn infants have a somewhat higher normal average. A neutrophilic band count greater than 11% is considered to be consistent with an inflammatory condition. In adults, the normal average for segmented neutrophils is 56% and approximately 4% for monocytes.