Hemoglobin

 
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Hemoglobin (Hgb), which consists of a heme group (porphyrin ring containing ferrous or Fe2+ iron) plus a pair of α and a pair of β globin chains, carries oxygen. In humans, hemoglobin is typically used to evaluate red blood cell mass versus PCV or HCT. However, in animals we generally default to the HCT or PCV because the hemoglobin can be falsely high in post-prandial samples from lipemia (see below), which we see more frequently than we would like in samples from dogs and cats. Since red blood cells are approximately 33% hemoglobin, the hemoglobin concentration of whole blood normally is about one third of the HCT (i.e., the MCHC is 33 g/dL). This is true of most species, other than camelids, whose hemoglobin takes up just less than half of the RBC (MCHC around 40-45 g/dL).

Method of measurement

The automated hematology analyzer at Cornell University provides two different measurement of hemoglobin. One is a direct spectrophotometric measurement using cyanide and the other is an optical measurement using laser light (calculated hemoglobin). These usually provide similar results in normal animals but differing results in animals with abnormal blood features, e.g. lipemia, agglutination. We usually do not provide results for calculated hemoglobin, unless we think the hemoglobin results by the regular spectrophotometric method are inaccurate. We have provided a table below for the different hemoglobin-related measurements that we can provide with our hemogram results (what they are, when we provide them).
CH

CH histogram

  • Red blood cell lysis and cyanmethemoglobin (measured hemoglobin): Traditionally, hemoglobin is measured using the cyanmethemoglobin method. To measure the hemoglobin concentration, a lysing agent is added to a sample of diluted blood; the lysing agent disrupts all the red blood cells in the sample and releases the hemoglobin into the fluid so that the sample then consists of a solution of hemoglobin. The hemoglobin is converted to a form called cyanmethemoglobin and the concentration is read by a spectrophotometer with the wavelength set at the peak absorbance of cyanmethemoglobin (540 nm). The concentration of hemoglobin is then calculated from the optical density of the solution. Conditions which cause turbidity in the lysate used in this assay, such as lipemia, Heinz bodies, or red blood cell nuclei (avian, reptilian blood) can result in falsely high absorbance and hence, overestimation of the hemoglobin concentration. Remember that hemoglobin-based oxygen carriers, like oxyglobin, are red and are measured as hemoglobin with the cyanmethemoglobin method. This will always result in a high hemoglobin concentrations (compared to the HCT and RBC count). With this technique, if the RBC are already lysed in the fluid, the hemoglobin will be the same (i.e. it does not matter if the RBC were prelysed with artifactual hemolysis or lysed by the added reagent). Thus, with in vitro hemolysis (an artifact of sample collection and handling), the measured hemoglobin concentration will be the most accurate result. With in vitro hemolysis, a HCT can be estimated from this hemoglobin measurement (by multiplying the hemoglobin x 3, because hemoglobin comprises approximately 1/3 of a RBC). The measured hemoglobin is used to calculate the following RBC indices, which are routinely provided on hemogram results:
    • MCH (pg): This is equivalent to (Hgb ÷ RBC count) x 10. Thus, MCH will be falsely increased if the Hgb is falsely increased or the RBC is falsely low (e.g. in vitro hemolysis).
    • MCHC (g/dL): This is equivalent to (Hgb ÷ HCT or PCV) x 100. The MCHC will be affected by the same things that affects the MCH, with the addition of the MCV (since HCT = MCV x RBC count).
  • Laser-based hemoglobin analysis

    Laser-based hemoglobin analysis

    Light scatter (intracellular hemoglobin): The hematology analyzer used at Cornell University (ADVIA 2120) also measures the hemoglobin content within all red blood cells directly, based on the internal complexity of the cells (which alters light scattering, creating high angle light scatter or side scatter). Based on the degree of light scatter, the analyzer can obtain a measure of the average hemoglobin content within all the red blood cells (called CHCM) or within individual red blood cells (called CH). These values can be more accurate than traditional methods of measuring hemoglobin using red blood cell lysis and cyanide, especially in conditions that falsely increase hemoglobin, such as oxyglobin or lipemia. The instrument also "channelizes" the scatter, segregating the cells into channels representing relative ranges of hemoglobin content. This is illustrated as a frequency distribution curve or histogram (see image above). The variation in hemoglobin content in intact red blood cells can also be calculated as the hemoglobin distribution width ( = standard deviation of the hemoglobin content ÷ mean hemoglobin content), however this value is not reported on hemograms (for internal use only). The ADVIA 2120 also converts the CHCM into a cellular hemoglobin measurement, which is a calculated value. We only use these results if we consider that the measured hemoglobin with the cyanide method is inaccurate (see above and table below).
    • CH: Directly measured by the high angle light scatter in the analyzer and is a measure of the amount of hemoglobin in each intact red blood cell (RBC must be intact to scatter the laser light, so prelysed RBC will not be detected with this method). A frequency distribution curve (histogram) of the variation in the hemoglobin content (CH) of intact RBC is also provided (see image above).
    • CHCM: Directly measured by the analyzer, like the CH.
    • Calculated hemoglobin: The ADVIA back-calculates a hemoglobin (calculated hemoglobin) from the CHCM (i.e. calculated hemoglobin = (CHCM x HCT) ÷ 1000 or (CHCM x MCV x RBC count) ÷ 1000. This provides reasonably accurate measurements of hemoglobin concentration in conditions which falsely increase the hemoglobin by the cyanmethemoglobin method (usually lipemia), however values will be falsely low with in vitro hemolysis (only the hemoglobin in unlysed RBC will be measured).
    • Free hemoglobin refers to the difference between the calculated and measured hemoglobin. The difference between these two measurements is normally quite small unless there is lipemia or hemolysis.

Units of measurement

The regular lysis hemoglobin or calculated hemoglobin are expressed as g/dL of the blood (SI units are g/L). The conversion formula to SI units is as follows

g/dL x 10 = g/L

 

Sample considerations

Sample type

Whole blood

Anticoagulant

EDTA is the preferred anticoagulant. Although citrate can be used, the volume of citrate in the tube (10% of the collection volume) will dilute the measured hemoglobin (but not the calculated hemoglobin) accordingly. Heparinized whole blood can also be used.

Stability

Hemoglobin is quite stable (the most stable of all RBC results).

Interferences

  • Lipemia: Will falsely increase measured hemoglobin and related calculated indices, MCH and MCHC.
  • Hemolysis: Will  decrease the calculated hemoglobin, CH and CHCM but has no effect on the measured hemoglobin. Hemolysis will also falsely increase the MCH (hemoglobin will be higher than RBC count) and MCHC (hemoglobin will be higher than HCT/PCV). In an animal with in vitro hemolysis versus true intravascular hemolysis, the measured hemoglobin is the best or most accurate estimate of the oxygen-carrying capacity of blood (multiply by 3 to obtain an approximate HCT). In contrast, the hematocrit (PCV), RBC count or calculated hemoglobin are a better estimate of the oxygen-carrying capacity of blood in an animal with true in vivo intravascular hemolysis (because the free hemoglobin in the animal's plasma from the intravascular hemolysis cannot carry oxygen, but is measured as hemoglobin by analyzers).
  • Icterus: No effect on either hemoglobin measurement.
  • OtherHeinz bodies (many, particularly if large) may falsely increase the measured hemoglobin. Oxyglobin will contribute to (falsely increase) the measured hemoglobin concentration.

Test interpretation

Increased concentration

  • Artifact: 
    • Measured hemoglobin: Lipemia, Heinz bodies, oxyhemoglobin, RBC nuclei (many nRBC).
  • Pathophysiologic
    • Relative change in RBC number to blood water: Dehydration, splenic contraction secondary to epinephrine (horses).
    • Absolute increase in RBC mass (erythrocytosis): Stimulated by erythropoietin (secondary erythrocytosis) or erythropoietin-independent (primary erythrocytosis, e.g. polycythemia vera or chronic myeloid leukemia)

Decreased concentration

  • Artifact: 
    • Calculated hemoglobin: Hemolysis of RBC due to sample collection or storage.
  • Pathophysiologic
    • Relative change of RBC to blood water: Over-dilution with fluids, splenic relaxation (anesthetic agents, tranquilizers).
    • Absolute decrease in RBC mass: Indicates a true anemia, due to hemorrhage, hemolysis (intravascular, extravascular) or decreased production. Multiple mechanisms may be operative.
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