RBCs are defined by three quantitative values: the volume of packed red cells or hematocrit (Hct), the amount of hemoglobin (Hb), and the red cell concentration per unit volume. Three additional indices describing average qualitative characteristics of the red cell polupation are also collected. These are mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).
Volume of Packed Red Cells (Hematocrit)
The hematocrit is the proportion of the volume of a blood sample that is occupied by red cells. Hct may be determined manually by centrifugation of blood at a given speed and time in a standardized glass tube with a uniform bore. The height of the column of red cells after centrifugation compared with total blood sample volume yields the Hct. However, several sources of error are inherent in the manual methods of measuring Hct technique. The spun Hct measures the red cell concentration, not red cell mass. Therefore, patients in shock or with volume depletion may have normal or high Hct measurements due to hemoconcentration despite a decreased red cell mass. In addition, technical sources of error in manual Hct determinations usually arise from inappropriate concentrations of anticoagulants, poor mixing of samples, or insufficient centrigugation. Another inherent error in manual Hct determinations arises from trapping of plasma in the red cell column. This may account for 1% to 3% of the volume in microcapillary tube methods, with macrotube methods trapping relative more plasma. It should be noted that abnormal red cells (e.g., sickle cells, microcytic cells, macrocytic cells, or spherocytes) often trap higher volumes of plasma due to increased cellular rigidity, possibly accounting for up to 6% of the red cell volume. Very high Hcts, as in polycythemia, may also have excess plasma trapping. Manual Hct methods typically have a precision coefficient of variation (CV) of approximately 2%.
Automated analyzers do not depend on centrifugation techniques to determine Hct, but instead calculate Hct by direct measurements of red cell number and red cell volume (Hct = red cell number X mean red cell volume). Automated Hct values closely parallel manually obtained measurements, and the manual Hct is used as the reference method for hematology analyzers (wtih correction for the error induced by plasma trapping). Errors of automated Hct calculation are more common in patients with polycythemia or abnormal plasma osmotic pressure. Manual methods of Hct determination may be preferable in these cases. The precision of most automated Hcts is <1% (CV).
Hemoglobin (Hb) is an intensely colored protein, allowing its measurement by spectrophotometric techniques. Hemoglobin is found in the blood in a variety of forms, including oxyhemoglobin, carboxyhemoglobin, methemoglobin, and other minor components. These may be converted to a single stable compound, cyanmethemoglobin, by mixing blood with Drabkin solution. Sulfhemoglobin is not converted but is rarely present in significant amounts. The main errors in measurement arise from dilution errors of increased sampel turbidity due to improperly lysed red cells, leukocytosis, or increased levels of lipid or protein in the plasma. With automated methods the precision for hemoglobin determinations is <1% (CV).
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 numbers. Both erythrocytes and leukocytes are counted after whole blood dilution in an isotonic solution. As the number of red cells greatly exceeds the number of white cells, the error introduced by counting both cell types is negligible. However, when marked keukocytosis is present, red cell counts and volume determinations may be erroneous unless corrected for white cells. The observed precision for RBC counts using automated hematology analyzers is <1% (CV) compared with a minimum estimated value of 11% with manual methods.
Mean Corpuscular Volume
The MCV is usually measured directly with automated instruments but may also be calculated from the erythrocyte count and the Hct by means of the following formula:
MCV = Hct (L/L) X 1,000/red cell count (1012/L)
The MCV is measured in femtoliters (fl, or 10-15 L). Using automated methods, this value is derived by dividing the summation of the red cell volumes by the erythrocyte count. The CV in most automated system is approximately 1%, compared to ~10% for manual method. Agglutination of cells, as in cold agglutinin disease or paraproteinemia, may result in a falsely elevated MCV. Most automated analyzers gate out MCV values above 360 fl, thereyby excluding most cell clumps, although this may falsely lower Hct determinations. In addition, severe hyperglycemia (glucose >600 mg/dL) may cause osmotic swelling of the red cells, leading to a falsely elevated MCV.
Mean Corpuscular Hemoglobin
MCH is a measure of the average hemoglobin content per red cell. It may be calcuated manually or by automated methods using the following formula:
MCH = hemoglobin (g/L)/red cell count (1012/L)
MCH is expressed in picograms (pg, or 10-12 g). Thus, the MCH is a reflection of hemoglobin mass. MCH measurements may be falsely elevated by hyperlipidemia, as increased plasma turbidity will erroneously elevate hemoglobin measurement. Centrifugaton of the blood sample to eliminate the turbidity followed by manual hemoglobin determination allows correction of the MCH value. Leukocytosis may also spuriously elevate MCH values. The CV for automated analysis of MCH is <1% in most modern analyzers, compared with approximately 10% for manual methods.
Mean Corpuscular Hemoglobin Concentration
The average concentration of hemoglobin in a given red cell volume or MCHC may be calcualted by the following formula:
MCHC = hemoglobin (g/dL)/Hct (L/L)
The MCHC is expressed in grams of hemoglobin per deciliter of packed RBCs, representing the ratio of hemoglobin mass to the volume of red cells. With the exception of hereditar 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, and further increases in Hb may lead to crystallization. Factors that alert the accuracy of both Hct and hemoglobin can affect the precision of MCHC.
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.
Determination of the numbers of reticulocytes or immature, non-nucleated RBCs that still retain RNA provides useful information about the bone marrow's capacity to synthesize and release red cells in response to a physiologic challenge, such as anemia. In the past, reticulocyte counts were performed manually using supravital staining with methylene blue that will stain precipitated RNA as a dark blue meshwork or granules (at least two per cell), allowing retriculocytes to be identified and enumerated manually. Because there are relatively low numbers of reticulocytes, the CV for reticulocyte counting is relatively large (10% to 20%).
To increase accuracy of reticulocyte counting, automated detection methods to detect staining allow for many more cells to be analyzed, thereby increasing accuracy and precision of counts. Most of the newest automated hematology analyzers have automated reticulocyte counts to be included with routine complete blood count parameters. Reticulocytes are detected by a fluorescent dye that binds to RNA. Comparisons of stand-alone instruments and integrated hematology analyzers demonstrate superior accuracy when compared to manual counting methods, with CVs of 5% to 8%.
Update on Aug 2nd 2017
Ontogeny of Hemoglobin
The hemoglobin composition of the erythrocyte depends on when in gestation or postnatal development it is measured. This is a result of sequential activation and inactivation (i.e., switching) among genes within the alpha- and non-alpha-globin gene clusters. What controls these switches in globin gene transcription is not understood. The two early embryonic hemoglobins consist of ζ- and ε-globin chains (Hb Gower-1) and α- and ε-globin chains (Hb Gower-2). The ζ-globin gene is akin to the α-globin genes but is expressed only during early embryogenesis. The ε-embryonic globin chain is a β-like element. The combination of ζ- and γ-globin chains forms hemoglobin Portland. These early hemoglobins are made primarily in yolk-sac erythroblasts and are detectable only during the very earliest stages of embryogenesis except in certain pathologic states, in which they may persist until gestation is complete. The major hemoglobin of intrauterine life is HbF, which consists of two α- and γ-globin chains. Expression of the γ-globin gene begins early in embryogenesis, peaks during midgestation, and begins a rapid decline just before birth. By 6 months of age in normal infants, only a remnant of prior γ-globin gene expression remains. The level of HbF in the blood declines rapidly thereafter to less than 1% of the total. Expression of the α-globin gene starts early in the first trimester, peaks quickly, and is sustained for life. Expression of the β-globin gene also commences early in gestation and reaches its zenith within a few months after birth. The combination of α-globin with β-globin cahins forms hemoglobin A (HbA), the predominant hemoglobin of postnatal life. Adult cells also contain HbA2. The δ-globin gene, which directs synthesis of the non-α-globin chain of HbA2, is very inefficiently expressed. Only low levels of HbA2 are present; defects in the δ-globin gene are of no clinical consequence. In adult blood, HbF is not evenly distribbuted among erythrocytes and is present in only a very small number of RBCs, called F cells. HbA2 is present in all RBCs, albeit at levels less than 3.5% of the total hemoglobin in adult life.