Routine Hematologic Examinations: Hemoglobin and Hematocrit Determination

Hematocrit (Hct) is defined as the volume of packed red blood cells (RBCs) that occupies a given volume of whole blood.
It represents the ratio of the volume of erythrocytes to the volume of the entire whole blood sample.
This value is frequently referred to as the packed cell volume (PCV).
Reporting units for hematocrit are typically expressed either as a percentage ( $\%$ ) or in liters per liter ( $L/L$ ).
Normal Reference Values:
- Men: $0.40 - 0.54$
- Women: $0.35 - 0.49$
- Newborns: $0.44 - 0.64$
Blood Composition and Clinical Status:
- Normal blood typically consists of plasma (water, proteins, nutrients, hormones), a buffy coat (white blood cells and platelets), and red blood cells.
- Normal hematocrit ranges indicated: $37\% - 47\%$ and $42\% - 52\%$ .
- Anemia: Characterized by a depressed hematocrit percentage.
- Polycythemia: Characterized by an elevated hematocrit percentage.

Macrohematocrit (Wintrobe Method):
- This method is now considered obsolete.
- Anticoagulants used include EDTA or ammonium-potassium oxalate.
- The Wintrobe tube has a length of $115\,mm$ and an internal bore of $3\,mm$ .
- It serves a dual purpose: reading both the Erythrocyte Sedimentation Rate (ESR) and the Macrohematocrit (MacroHct).
- Scaling of the Wintrobe tube:
  - ESR (left side): $0$ at the top to $100$ at the bottom.
  - MacroHct (right side): $100$ at the top to $0$ at the bottom.
Microhematocrit (Adam’s Method):
- This method utilizes fresh, well-mixed blood anticoagulated with EDTA.
- Two capillary tubes are filled approximately three-fourths ( $3/4$ ) full with the blood sample.
- The tubes are centrifuged at speeds ranging from $10,000\,g$ to $15,000\,g$ .

Capillary Tube Types:
- Red-Banded Capillary Tube: Contains Heparin; used to collect blood directly from a skin puncture.
- Blue-Banded Capillary Tube: Contains no anticoagulant; used to collect blood from an existing EDTA tube.
Sources of False Increases in Hematocrit:
- Dehydration.
- Hemoconcentration.
- Insufficient centrifugation.
- Inclusion of the buffy coat when measuring the red cell column.
- Prolonged application of a tourniquet during venipuncture.
Sources of False Decreases in Hematocrit:
- Poikilocytes (abnormally shaped RBCs).
- Hemolysis of red blood cells.
- Improper sealing of the capillary tube.
- Decreased anticoagulant concentration leading to RBC shrinkage.
- Introduction of interstitial fluid into the sample.
Trapped Plasma:
- This phenomenon causes manual microhematocrit readings to be $1\%$ to $3\%$ higher than automated hematocrit results.
- Conditions associated with trapped plasma include:
  - Sickle Cell Anemia.
  - Hypochromic Anemia.
  - Spherocytosis.
  - Macrocytosis.
  - Thalassemia.

The reference method for hemoglobin measurement is the Cyanmethemoglobin (HiCN) method.
Principle: The absorbance of the solution at $540\,nm$ is directly proportional to the concentration of hemoglobin.
Advantages of Cyanmethemoglobin Method:
- Drabkin’s reagent is highly stable.
- The method can measure all forms of hemoglobin with the exception of sulfhemoglobin.
Normal Hemoglobin Reference Values:
- Men: $13.5 - 18.0\,g/dL$
- Women: $12.0 - 15.0\,g/dL$

Technical Errors in Cyanmethemoglobin Method:
- Light Sensitivity: The reagent is sensitive to light and must be stored in dark places or brown bottles.
- High Cell Counts: A high WBC count ( $> 20 \times 10^9/L$ ) or high platelet count ( $> 700 \times 10^9/L$ ) causes turbidity and false high results. Correction involves centrifuging the reagent-sample solution and measuring the supernatant.
Physiologic Errors and Corrections:
- Lipemia: To correct for lipemia, add $0.01\,mL$ of patient plasma to $5.0\,mL$ of cyanmethemoglobin reagent to create a sample blank.
- Extremely High WBC Count ( $> 30 \times 10^9/L$ ): Correction requires centrifuging the mixture and determining the hemoglobin concentration using the supernatant.
- Hemoglobin S (Hb S) and Hemoglobin C (Hb C): These can cause turbidity. Correction involves diluting the mixture by a ratio of $1:2$ (one part sample mixed with one part distilled water) and multiplying the resulting absorbance by $2$ .

Gasometric Method:
- This measures the oxygen-carrying capacity of the blood using a Vonslyke apparatus.
- Hemoglobin is estimated indirectly based on the assumption that $1\,g$ of hemoglobin carries $1.34\,mL$ of oxygen.
Specific Gravity Method:
- Utilizes a copper sulfate ( $CuSO_4$ ) solution.
- This is routinely used to screen blood donors for anemia.
- A copper sulfate solution with a specific gravity of $1.053$ is considered equivalent to $12.5\,g\%$ of hemoglobin.
Chemical Method:
- An indirect method based on the assumption that $0.347\,mg$ of iron is compared with $100\,g$ of hemoglobin.

SYSMEX Autoanalyzers:
- Principle: Spectrophotometry (colorimetry).
- Blood is diluted and mixed with a sodium lauryl sulphate (SLS) hemoglobin reagent.
- SLS converts ferrous iron ( $Fe^{2+}$ ) to ferric iron ( $Fe^{3+}$ ) to form methemoglobin.
- The absorbance is measured at a wavelength of $555\,nm$ .
HEMOCUE System:
- Blood is drawn by capillary action into a cuvette containing sodium nitrate and sodium azide.
- These reagents convert hemoglobin into azide methemoglobin.
- The absorbance of the resulting azide methemoglobin is measured at a wavelength of $565\,nm$ .