CBC Lecture Notes

Complete Blood Count (CBC)

Learning Objectives

Explain the Complete Blood Count (CBC) test.
Identify the correct specimen and collection requirements for the CBC test.
Discuss specimen criteria for rejection and follow-up procedures.
List the component tests that make up the CBC.
Explain what each component is measuring.
State normal reference ranges for all components of the CBC test.
Explain the principle of common automated CBC analyzers.
Recognize appropriate action for specimen integrity issues (e.g., hemolysis, icteric, lipemic, clotted, low volume, etc.).
Describe storage requirements for CBC blood tubes used in testing.

Required Reading

Turgeon, M.L. (Last Published). Linné & Ringsrud’s Clinical Laboratory Science, Concepts, Procedures and Clinical Applications (Current Ed.). Elsevier Health Sciences.
Chapter 11 – Pages 308-310, 312-319, 320-322

CBC Specimen Blood Collection

EDTA Tube:
- A 4 mL tube is most common for CBC tests.
- Ideally filled to the top, needing a minimum of 1 mL to run the test.
- Underfilling alters the anticoagulant-to-blood ratio, potentially changing the morphology of red cells.
- Proper mixing after collection prevents clotting.
- Collect using the CLSI order of draw.
- Test as soon as possible for optimal accuracy.
- If refrigerated, allow to warm up at room temperature for about 30 minutes before testing.

CBC Specimen Blood Collection Processing and Testing

When a collected specimen stands for a time, the components settle into three distinct layers:
- Top layer: plasma
- Middle layer: buffy coat, a grayish-white cellular layer composed of WBCs and platelets
- Bottom layer: RBCs
Appearance of specimens (look for clots!)
Hemolysis (what would this mean? – in vivo or in vitro?)
Unsuitable hematologic specimens (wrong tube, low volume…?).

Automated CBC Analyzer

Used to manually count (Hemocytometer) until 1950s
Automated counter (1956)
- Principle of electrical impedance.
- Used to just count RBCs and WBCs.
Basic Principles to CBC Analyzers:
- Electrical impedance counting.
- Light scatter (optical detection).

CBC – Complete Blood Count

Laboratory tests performed in the hematology laboratory include the following:
- Counting the number or concentration of cells
- Determining the relative distribution of various types of cells
Tests to help diagnose and monitor treatment:
- Complete blood count (CBC): Hemoglobin (Hb), hematocrit (Hct), RBC count with morphology, WBC count with differential, and platelet estimate.
- The RBC indices (calculations) of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) are now a standard part of a routine automated CBC.
Blood cell counts
- Counting the various cells (RBC, WBC, PLT) found in blood is a fundamental procedure in the hematology laboratory.
- Electronic counting devices avoid human error, which is significant in manual cell counts, and are statistically more accurate because of sampling; these devices count many more cells than can be counted manually.

Modes of Operation

Primary mode (automatic)
- Multiple samples are mixed, aspirated, and analyzed automatically from the primary tubes (closed system).
- Probe has cap piercing capability
Secondary mode (manual)
- Samples are mixed manually, and caps are removed.
- Patient information and tests are entered manually.
- Each sample is run individually via the secondary aspiration mode

Hematology Procedures – Tonicity

When in plasma, the red blood cells are in an isotonic solution.
Any diluent used to dilute blood for CBC testing must have the same ionic concentration as plasma; then the solution is called a physiologic solution (0.85% Saline).
Saline is a sodium chloride (NaCl) solution.
NOTE: A comparison of erythrocytes in three concentrations of sodium chloride solution demonstrates the net movement of water molecules into and out of the cell.
- Hypertonic - If the net movement of water out of the cell is in excess, the cell will crenate (shrinks & wrinkles)
- Hypotonic - If the net movement of water into the cell is in excess, the cell swells up and will lyse or burst.
- Isotonic – no movement, cells remain the same

Principles of Automation

Electrical impedance (counting)
Optical/Light scattering (optical detection)

Electrical Impedance

Coulter Principle of Cell Counting
- Cells pass through an aperture, which current is flowing, and cause changes in electrical resistance that are counted as voltage pulses
- A vacuum draws the cell suspension through the aperture
- Each cell that passes, increases electrical resistance and creates a pulse, each pulse is counted
- Size of pulse is directly proportional to size of cell
- Histograms are generated by the pulses
- COINCIDENCE ERROR: when two cells go through together, counted as one
Volume Distribution Histograms
- The number of pulses generated is directly proportional to the number of cells being counted.
- The height of the pulse is directly proportional to the size of the cells passing through the aperture.
- Three histograms can be generated:
  - WBC
  - RBC
  - PLT
RBC and WBC baths on the Beckman Coulter Blood Analyzer Single aperture system (3 separates counts)
- In this case the RBC and Platelets are counted in the RBC bath on the left.
- The WBC’s are counted and sized on in the WBC bath on the right
- Hemoglobin measurement is done in the WBC bath using Cyanmethemoglobin method, RBCs are lysed and Hgb released

Optical (Light) Scatter Principle

Also known as flow cytometers
Measures light scatter (forward and side)
May be used as a primary method or in combination with others
Uses a flow tube to stream fluid through a quartz flow cell and passes through a light source (tungsten-halogen lamp or helium-neon laser)
Interruption of the beam of light occurs when the cells pass through the sensing zone
The light “scatters” as it comes in contact with cells and can be measured at multiple angles
Light is scattered in all directions as the cell interrupts the light source.
As light is scattered, it is converted to an electrical signal when it comes in contact with the photodetector.
Filters and mirrors separate the different wavelengths
Flow Cytometry: Optical (light) scatter
- Forward-angle light scatter – 0 degrees – correlates with cell volume, mainly due to the diffraction of light.
- Side light scatter – 90 degrees – measure large structures inside the cell and correlates with internal cellular complexity.
- Scatter measurements combine to identify the different types of WBCs, and each cell population is demonstrated/plotted on a Scatter Plot diagram

Hemoglobin

Hemoglobin measurement:
In a Coulter Analyzer: HGB determination (Cyanmethemoglobin method), is performed in the WBC bath using Drabkin’s reagent, following the lysing of the RBC and release of HGB.
To avoid danger of using cyanide – other analyzers (i.e., Sysmex) are using cyanide-free Sodium Lauryl Sulfate (SLS) reagent to lyse RBC’s and WBC’s in the sample
- Alters globin chains, exposing the heme group.
- The heme group is then oxidized allowing the SLS reagent to form a stable colored complex with the Heme portion
- Analyzed via a photometric method where the amount of light absorbed (absorbance) is proportional to the hemoglobin concentration of the sample.

What is the CBC Counting?

Blood cell counts:
- Red Blood Cell count
- White Blood Cell count
- Platelet count

Red Blood Cell Parameters

RBC Count x 10^{12}/L
HGB (g/L)
HCT (L/L or %)
Mean cell volume/MCV (fL)
Mean cell hemoglobin/MCH (pg)
Mean cell hemoglobin concentration/MCHC (g/L)
RBC Distribution Width/RDW (%)
Morphology RBC Calculated Indices

Red Blood Cell Count – # of RBCs
Hematocrit – packed cell volume (PCV)
Hemoglobin – protein that carries O_2 to tissues
RBC Indices

Red Blood Cell Indices

Mean corpuscular volume (MCV) is the average volume of an RBC in femtoliters, as calculated in this equation:
MCV (fL) = \frac{Hct × 10}{RBC}
where MCV is mean corpuscular volume, fL is femtoliters, Hct is hematocrit, and RBC is red blood cell count.
Reference range is 80 to 96 fL.
Mean corpuscular hemoglobin (MCH) is the content (weight) of hemoglobin in the average RBC, as calculated in this equation:
MCH (pg) = \frac{Hb × 10}{RBC}
where MCH is mean corpuscular hemoglobin, pg is picograms, and Hb is hemoglobin.
Reference range is 27 to 33 pg.
Mean corpuscular hemoglobin concentration (MCHC) is the average Hb concentration in a given volume of packed RBCs, as calculated with the equation:
MCHC (g/dL) = \frac{MCH}{MCV} × 100
where MCHC is mean corpuscular hemoglobin concentration, g is grams, and dL is deciliters.
Reference range is 33 to 36 g/dL.
MCV = HCT / RBC
- RI: 80-100 fL
MCH = HGB / RBC
- RI: 26-34 pg
MCHC = HGB / HCT
- RI: 320-360 g/L
Red cell distribution width
- Red cell distribution width (RDW) is a measurement of the degree of anisocytosis present, or the degree of variability in RBC size, in a blood specimen, as shown in this calculation:
  RDW (%) = \frac{Standard \ deviation \ (SD) \ of \ MCV}{Mean \ MCV} × 100
  Reference range is 11% to 15%.

CBC- Reticulocytes

Reticulocyte counts
- Reticulocytes are red blood cells that have lost their nuclei but not all of their cytoplasmic RNA.
- Normal erythropoiesis and reticulocytes
- High reticulocyte count, reticulocytosis, is a clinical indication that the body is attempting to meet an increased need for RBCs.
- Clinical uses for reticulocyte counts
  - Follow therapy for anemia
- Reference values
  - Adults: 0.5% to 1.5% of circulating red blood cells
  - Newborn: 2.5% to 6.0%

WBC - Leukocytes

Reporting leukocyte results
- Total count
  - The total leukocytes in the circulating blood vary by age.
  - Can fluctuate with circadian rhythms
- Relative count
  - In the differential leukocyte count, cells are identified while examining and counting 100 WBCs in a systematic manner, with results reported in relative numbers or percentages.
- Absolute count
  - The absolute count is a more accurate measure. The absolute cell count by cell type is obtained by multiplying the relative number of WBCs (in decimal units) by the total WBC count per liter.

White Blood Cell Differential

Identifies and counts the different kinds of white blood cells
- Neutrophils
- Lymphocytes
- Monocytes
- Eosinophils
- Basophils

CBC Analyzer Reference Ranges

RBC (adult male): 4.5-6.5 \times 10^{12}/L
RBC (adult female): 3.8-5.8 \times 10^{12}/L
Hemoglobin (adult male): 140-180 g/L (Critical value: <70)
Hemoglobin (adult female): 120-160 g/L (Critical value: <70)
Hematocrit (adult male): 0.42-0.54 L/L or %
Hematocrit (adult female): 0.37-0.47 L/L or %
Mean Cell Volume (MCV): 80-100 fL
Mean Cell Hemoglobin (MCH): 27-31 pg
Mean Cell Hemoglobin Concentration (MCHC): 320-360 g/L
Reticulocyte Count: 0.5-2.0 Relative %
Red Cell Distribution Width (RDW): 11.5-14.5 %
Nucleated RBC (NRBC): 0 \times 10^9/L
WBC: 4.0-11.0 \times 10^9/L (Critical values:
Neutrophils: 48-70 % (relative)
Lymphocytes: 18-42 % (relative)
Monocytes: 1-10 % (relative)
Eosinophils: 1-4 % (relative)
Basophils: 0-2 % (relative)
Platelet Count (PLT): 150-400 \times 10^9/L (Critical values:
Mean Platelet Volume (MPV): 9-12.5 fL

Specimen Integrity Issues for EDTA

Read label to see if CBC test ordered.
Correct tube (4mL or 6mL unspun EDTA).
Low volume (insufficient quantity) – may still be able to run, check for clots first; can affect the morphology of the cells.
Clotted specimens – if clots seen, do NOT run.
Hemolyzed specimens – if from mishandled specimens, RBC count would be falsely decreased.
Lipemic specimens – a high MCHC would suggest the presence of an interfering specimen like lipids.

Storage Requirements for EDTA Tubes

Run collected samples as soon as possible if kept at room temperature.
Samples can be stored refrigerated (4°C) up to 12 hours before running.
Return to room temperature before running.
After testing, samples are stored in the fridge for one week
After one week, samples can be discarded appropriately in the yellow biohazard buckets

CBC Analyzer Daily Maintenance

Check reagents (lot #’s, expiry dates, and levels)
Check printer and paper (replace paper if needed)
Ensure the racks are clean and good to use
Clean probes
Empty the waste container
Wipe down analyzer with distilled water (keeps dust down)
Run daily controls (follow lab protocol and manufacturers recommendations)

CBC Quality Controls

3 levels of controls are run at the start of each shift (every 8 hours)
Monitor all CBC parameters tested on the analyzer (low, med, high)
Assumed that when controls are within expected reference ranges, the analyzer is producing accurate and precise results
Controls are human blood (same matrix as samples) and must be used following Routine Practices
Check the lot # and expiry of the control material
Controls are stored in the fridge and must be warmed up for 15-30 minutes prior to analysis.
Mix the vials gently (end-to-end) and ensure the cell button is completely off the bottom of the tube before running
If the controls are out of the established range, then further action is required before running patient samples.