|Parameters measured by most analyzers||Parameters measured by some analyzers|
|Parameters measured directly or derived through histogram||Parameters measured through calculation|
Estimation of Hemoglobin
Hemoglobin is measured directly by a modification of cyanmethemoglobin method (all hemoglobins are converted to cyanmethemoglobin by potassium ferricyanide; cyanmethemoglobin has a broad absorbance peak at 540 nm). Some analyzers use a nonhazardous reagent such as sodium lauryl sulphate. A non-ionic detergent is added for rapid red cell lysis and to minimize turbidity caused by cell membranes and plasma lipids.
Estimation of Red Blood Cell Count and Mean Cell Volume (MCV)
Red cell count and cell volume are directly measured by aperture impedance or light scatter analysis. In a red cell histogram, cell numbers are plotted on Y-axis, while cell volume is indicated on Xaxis (see Figure 808.1). The analyzer counts those cells as red cells volume of which ranges between 36 fl and 360 fl. MCV is used for morphological classification of anemia into microcytic, macrocytic, and normocytic types.
Estimation of MCH, MCHC, and Hematocrit (HCT/PCV)
These parameters are obtained indirectly through calculations.
MCH (pg) = Hemoglobin (g/l) ÷ RBC count (10⁶/μl)
MCHC (g/dl) = Hemoglobin (g/dl) ÷ Hematocrit (%)
Hematocrit (%) = Mean Cell Volume (fl) ÷ RBC count (10⁶/μl)
Estimation of Red Cell Distribution Width (RDW)
RDW is a quantitative measure of variation in sizes of red cells and is expressed as coefficient of variation of red cell size distribution. It is equivalent to anisocytosis observed on blood smear. It is derived from red cell histogram in some analyzers. RDW is usually elevated in iron deficiency anemia, but not in β-thalassemia minor and anemia of chronic disease (other causes of microcytic anemia). However, this distinction is not absolute and there is a significant overlap between values among patients. Raised RDW requires examination of blood smear.
Among the red cell values generated by the analyzer (red cell count, hemoglobin, hematocrit, MCV, MCH, MCHC, and RDW), most important for decision-making are hemoglobin, hematocrit, and MCV.
Difference between 3-part and 5-part hemotology analyzer...
Hematology analyzers can either generate a 3-part differential (differential count reported as lymphocytes, monocytes, and granulocytes) or a 5-part differential (lymphocytes, monocytes, neutrophils, eosinophils, and basophils). The 3-part differential counting is based on electrical impedance volume measurement of leukocytes. In volume histogram for WBCs, approximate numbers of cells are plotted on Y-axis and cell size on X-axis. Those cells with volume 35-90 fl are designated as lymphocytes, cells with volume 90-160 fl as mononuclear cells, and cells with volume 160-450 fl as neutrophils (see Figure 808.2). Any deviation from the expected histogram is flagged by the analyzer, mandating review of blood smear. A large proportion of 3-part differential counts are ‘flagged’ to avoid missing abnormal cells.
Instruments measuring a 5-part differential work on a combination of different principles, e.g. light scatter, impedance, and electrical conductivity, a combination of light scatter, peroxidase staining, and resistance of basophils to lysis in acid buffer, etc.
Platelets are difficult to count because of their small size, marked variation in size, tendency to aggregation, and overlapping of size with microcytic red cells, cellular fragments, and other debris. In hematology analyzers, this difficulty is addressed by mathematical analysis of platelet volume distribution so that it corresponds to lognormal distribution. Platelets are counted by electrical impedance method in the RBC aperture, and a histogram is generated with platelet volume on X-axis and relative cell frequency on Y-axis (see Figure 808.3). Normal platelet histogram consists of a right-skewed single peak. Particles greater than 2 fl and less than 20 fl are classified as platelets by the analyzer.
Two other platelet parameters can be obtained from platelet histogram using computer technology: mean platelet volume (MPV) and platelet distribution width (PDW). Some analyzers can generate another parameter called as reticulated platelets.
MPV refers to the average size of platelets and is obtained from mathematical calculation. Normal MPV is 7-10 fl. Increased MPV (> 10 fl) results from presence of immature platelets in circulation; peripheral destruction of platelets stimulates megakaryocytes to produce such platelets (e.g. in idiopathic thrombocytopenic purpura). Decreased MPV (< 7 fl) is due to presence of small platelets in circulation (in conditions associated with reduced production of platelets in bone marrow).
PDW is analogous to RDW and is a measure of variation in size of platelets (normal <20%). Increased PDW is observed in megaloblastic anemia, chronic myeloid leukemia, and after chemotherapy.
Some analyzers measure reticulated platelets or young platelets that contain RNA (similar to reticulocytes). Increased numbers of reticulated platelets are seen in thrombocytopenia due to peripheral destruction of platelets.
Various fluorescent dyes can combine with RNA of reticulocytes; the fluorescence then is counted in a flow cytometer. More immature reticulocytes fluoresce more strongly as they contain more RNA.
Reticulocyte hemoglobin content is a parameter that estimates hemoglobinization of most recently produced red cells. It is a predictor of iron deficiency.
WBC Cytogram (Scattergram)
In the scattergram, each dot represents a cell of a given volume and density, and the positions of dots in the graph are determined by the degree of side scatter, degree of forward scatter, light absorption by the cell, and cytochemical staining (if used). The forward angle light scatter (FALS) is represented on Y-axis, and the side scatter (SS) is represented on X-axis. Low FALS and low SS are indicative of lymphocytes; with subsequent increasing FALS and SS, monocytes, neutrophils, and lastly eosinophils are designated in the graph. Counting of basophils is based on a different technology.