hgb hct

• Hemoglobin is ordered by physicians to diagnose and monitor the course of anemia
• Hematologic analyzers include hemoglobin determination as a standard test in CBC
• Functions of hemoglobin:

  • Main component of red blood cells
  • Transports oxygen and carbon dioxide
  • Each gram of hemoglobin holds 1.34 mL of oxygen when fully saturated
  • Involved in acid-base balance
  • Responsible for binding, inactivation, and transport of nitric oxide (NO)
• Structure of hemoglobin:

  • Composed of two different pairs of polypeptide chains called Globin and four Heme groups
  • Each polypeptide chain has one embedded heme group
  • Hemoglobin is made up of 4 prosthetic heme groups
  • Lack of iron leads to insufficient hemoglobin formation and anemia
• Heme structure:

  1. Heme:

  • Protoporphyrin IX
  • Iron atom in the ferrous form (Fe2+)

  2. Globin:

  • Heme consists of a ring of carbon, hydrogen, and nitrogen atoms called Protoporphyrin IX with a divalent Ferrous Iron (Fe2+)
  • Ferrous iron can reversibly combine with one oxygen molecule
  • Each heme is precisely located in a pocket or fold of one of the polypeptide chains
  • Hemoglobin is made up of 4 prosthetic heme groups
• Oxyhemoglobin:

  • Ferrous (Fe2+) reversibly bound to oxygen
  • Relaxed form (with 2,3-BPG)
  • In the lungs, oxygen tension is high, allowing hemoglobin to bind with four oxygen molecules
• Deoxyhemoglobin:

  • Ferrous (Fe2+) not bound to oxygen
  • Tense form (with 2,3-BPG)
  • In the tissues, oxygen tension is low, leading to the dissociation of oxygen from hemoglobin
• Globin structure:

  • Hemoglobin is primarily made up of protein
  • Consists of two chain combinations of alpha, beta, delta, epsilon, gamma, or zeta
  • Normal production of these globin chains is essential for proper hemoglobin formation
• Dyshemoglobin:

  • Also known as dysfunction hemoglobins
  • Unable to transport oxygen
  • Can form and accumulate to toxic levels after exposure to certain triggers
  • These stimuli changes the hemoglobin structure, inhibiting the hemoglobin molecule from binding with oxygen
  • Severe impairment can result in hypoxia or cyanosis
  • If not corrected, it will eventually lead to death
  • Most cases are acquired; only a small fraction is hereditary
• Methemoglobin (Hi):

  • Normally 1-2%
  • Also known as Ferrihemoglobin or Hemiglobin
  • Formed by the reversible oxidation of Ferrous (Fe2+) to Ferric (Fe3+); cannot carry O2
  • Accumulation is prevented by methemoglobin reduction systems
  • Increased methemoglobin leads to decreased oxygen delivery to tissues
  • Color of blood (methemoglobinemia): chocolate brown
  • Most cases of methemoglobinemia are acquired, resulting from exposure to certain drugs and chemicals such as nitrates, nitrites, quinolones, and chlorates
• Carboxyhemoglobin (HbCO):

  • CO (carbon monoxide) bound to heme
  • Carbon monoxide has 210 times greater affinity to hemoglobin than O2
  • Silent killer; a tasteless, colorless, and odorless gas that can quickly make victims hypoxic
  • Binding is reversible
  • Treatment for carbon monoxide poisoning is the use of hyperbaric oxygen to remove carbon monoxide in the blood
  • Color of blood and skin in HbCO poisoning: cherry red
• Sulfhemoglobin (SHb):

  • Normally <1%
  • Sulfur bound to heme (greenish pigment)
  • Mixture of oxidized, partially denatured forms of hemoglobin
  • Irreversible (persists for the life of the cell)
  • Color of blood (sulfhemoglobinemia): mauve-lavender
  • Sulfhemoglobin is ineffective for oxygen transport, and patients with elevated levels present with cyanosis
  • Unlike methemoglobin, sulfhemoglobin cannot be reverted back to normal hemoglobin, and it remains in the cell for its life
• Hemoglobin Measurement in the Laboratory:

  1. Specific Gravity Method (Copper Sulfate Method):

  • Based on the estimation of the specific gravity of blood
  • A blood droplet, allowed to fall into a CuSO4 solution with a specific gravity of 1.053, becomes encased in a sac of copper proteinat, which prevents dispersion of fluid for 15 seconds
  • Specific Gravity of 1.05
• Copper proteinate prevents dispersion of fluid for 15 seconds
• Specific Gravity (SG) of 1.053 = 12.5 g/dL of hemoglobin
• Sample: Capillary blood
• Copper Sulfate Method Procedure:

  • Prepare the CuSO4 solution (SG = 1.053) in a wide-mouth container with 5-inch depth
  • Perform skin puncture
  • Allow the drop of blood to fall into the CuSO4 solution
  • Observe the movement of droplet for at least 15 seconds
  • Interpretation:
  • SG of blood > solution = Sink
  • SG of blood < solution = Float
• Acid Hematin Method (Sahli-Hellige Method):

  • Blood + 0.1 N HCl = Hemoglobin → Acid hematin (dark brown colored compound)
  • Dilute the solution with distilled water until its color intensity matches with the standard brown colored glass of the comparator box
  • The concentration of hemoglobin is read directly on the apparatus
  • Sample: EDTA whole blood
  • Procedure:
  • Prepare the materials
  • Add 0.1 N HCl up to the 2 mark on the hemoglobinometer
  • Pipette blood up to the 20th mark (equivalent to 0.02 mL or 20 uL blood)
  • Add the blood into the hemoglobinometer containing HCl
• • Rinse the contents of the pipette by drawing in and blowing out the acid 2-3 times
  • Thoroughly mix the blood with the acid
  • Allow to stand undisturbed for 10 mins
  • Place the hemoglobinometer in the comparator and add distilled water to the solution dropwise, stirring with the glass rod until its color matches with that of the comparator glass
  • Remove the glass rod and take the reading directly by noting the height of the solution
  • Report results in grams per 100 mL of blood (g/dL)
• • Sodium bicarbonate: shortens the time needed for the complete conversion of Hb to HCN (10 minutes); enhances lysis of erythrocytes and decreased turbidity from protein precipitation
  • Replaced by Dihydrogen potassium phosphate in the modified Drabkin's reagent: decreases reaction time to 3 mins only
  • Procedure:
  • Prepare all equipment & label tubes as: blank, standard, and unknown
  • Turn on the spectrophotometer and set the wavelength at 540 nm
  • Pipette as follows:
  • Dispense 5 mL of Drabkin's reagent into blank and unknown test tubes
• Cyanmethemoglobin Method (Hemiglobin Cyanide Method):

  • Potassium: Blood is diluted with Drabkin's reagent
  • Potassium ferricyanide oxidizes hemoglobin to methemoglobin
  • Potassium cyanide provides cyanide ions to form cyanmethemoglobin
  • The solution is read at 540mm wavelength and compared with that of a standard HCN solution
  • Sample: EDTA whole blood (0.02 mL sample + 5 mL reagent)
  • Drabkin’s Reagent:
  • Potassium ferricyanide: oxidizes ferrous iron to ferric state
  • Potassium cyanide: donates cyanide ions
• • Dispense 5 mL of hemoglobin standard into the standard test tube
  • Using a micropipette, draw 20 uL of blood, wipe excess blood from the exterior of the pipette, and dispense the blood into the unknown test tube
• Transfer the contents of the tubes into cuvettes
• Run the "blank" into the spectrophotometer and read absorbance
• Place the standard into the cuvette and read absorbance
• Run the patient's sample and read absorbance
• Calculate hemoglobin concentration using the formula
• Discard all specimens and contaminated materials into biohazard container
• Disinfect and clean equipment and return to proper storage
• Turbidity brought by:

  • WBC count (>20 x 10^9/L)
  • Platelet count (>700 x 10^9/L)
  • Centrifuge reagent-sample solution, then the supernatant is measured
  • Lipemia
  • Add 0.01mL of the patient's plasma to 5mL of the Drabkin's reagent and use this solution as the reagent blank
  • Cells containing Hb S and Hb C (resistant to lysis = turbidity)
  • Make a 1:2 dilution with distilled water (1 part sample plus 1 part water) and multiply the results from the standard curve by 2
• Errors inherent in the sample:

  • Improper venipuncture technique may introduce hemoconcentration
  • Hb CO (Carboxyhemoglobin) takes about 1 hour to convert to HiCN and theoretically could cause erroneous results in specimens from heavy smokers
• Errors inherent in the method:

  • Use of the HiCN standard for calibration of the instrument and for the test itself eliminates major source of error
  • The broad absorption band of HiCN in the region of 540 nm makes it convenient to use both in filter-type and narrow-band spectrophotometers
  • With the exception of SHb, all other varieties of hemoglobin are converted to HiCN
  • Cyanmethemoglobin reagent is sensitive to light
  • It should be stored in a brown bottle or in a dark place
• Errors inherent in the equipment:

  • The accuracy of equipment is not uniform
  • Calibration of pipettes will lessen errors
  • Significant error can be introduced by the use of unmatched cuvettes
  • The wavelength setting, the filters, and the meter readings require checking
• Operator's error reduced by:

  • Good training
  • An understanding of the clinical significance of the test
  • The necessity for a dependable method
  • Adherence to oral and written instructions
  • Familiarity with the equipment and with the sources of error
• Reference ranges (Henry's):

  • Male: 13.5 - 18 g/dL
  • Female: 12 - 16 g/dL
• Hematocrit:

  • The volume of packed RBCs that occupies a given volume of whole blood
  • Also known as Packed Cell Volume (PCV)
  • It is reported either as a percentage (%) or in liters per liter (L/L)
  • Suitable anticoagulants: Dried Heparin, Ethylenediaminetetraacetic acid (EDTA)
  • The hematocrit, or packed cell volume, is a macroscopic observation of the volume of packed red blood cells in a sample of whole blood, if measured by manual technique
  • It may be performed separately or as part of a complete blood count (CBC)
• • Hematocrit is defined as the ratio of the volume of erythrocytes to that of the whole blood
  • It may be expressed in conventional unit as percentage (%) or in SI unit as a decimal fraction indicated in liters per liter (L/L)
• Dried heparin and ethylenediaminetetraacetic acid (EDTA) are suitable anticoagulants
• Hematocrit may be measured directly by centrifugation with macromethods or micromethods, or indirectly as the product of the mean cell volume times the erythrocyte count in automated methods
• • Cloudy plasma may be suggestive of recent fat-rich meal, nephrosis (if blood is not collected within 1-2 hours after a meal), or presence of abnormal proteins (e.g., hyperglobulinemia, cryoglobulinemia)