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How analyzers count cells and measure hemoglobin

13 min

Skills you'll build3 skills
  • Explain how an impedance or optical analyzer counts cells and what it can miscount
  • Recognize and correct turbidity that falsely raises a photometric hemoglobin
  • Recognize trapped plasma, sealing, and spinning errors in a microhematocrit

Try first

Try first

An impedance analyzer sizes each cell by the height of the voltage pulse it produces as it crosses a narrow aperture. Two red cells cross the aperture together, at the same moment. What does the analyzer do with them?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Sizing cells by an electrical pulse

Most hematology analyzers count and size cells by electrical impedance, the Coulter principle. Cells suspended in a conductive diluent are drawn one at a time through a narrow aperture between two electrodes carrying a steady current. Each cell briefly raises the resistance across the aperture and produces a voltage pulse. The number of pulses gives the cell count, and the height of each pulse gives the cell's volume.1 Hydrodynamic focusing centers each cell in the stream before it reaches the aperture, which keeps the pulse height close to what the cell's true volume would produce.

Coincidence and autoagglutination

Two cells crossing the aperture at the same moment generate a single, oversized pulse. The count is falsely low and the volume histogram is shifted high, because the instrument counts one large event for two ordinary cells. Red-cell autoagglutination, most often from cold agglutinins, produces this same pattern across a whole specimen: fewer, larger apparent cells, a falsely low red-cell count, and a falsely high MCV and MCHC. Warming the specimen to 37 °C and rerunning it promptly disperses the agglutinates before the repeat count.4

Turbidity and the hemoglobin measurement

Hemoglobin is measured a different way, usually by a photometric reading of the lysed specimen at a fixed wavelength. Lipemia, a very high leukocyte or platelet count, and precipitated paraproteins each add turbidity, and turbidity raises absorbance at that wavelength the same way a true rise in hemoglobin would.3,4 The raised hemoglobin then raises the calculated MCH and MCHC. The red-cell count and the hematocrit calculated from it can stay close to the patient's baseline. The rule of three (hematocrit about 3 times the hemoglobin) detects this kind of mismatch before a result is released.4

Follow the bench demonstration

Sources for this step
References
  1. Keohane EM, Preston MM, Mirza KM, Walenga JM, eds. Rodak's Hematology: Clinical Principles and Applications. 7th ed. Elsevier; 2025. Accessed September 26, 2026. https://www.us.elsevierhealth.com/rodaks-hematology-9780323936507.html
  2. Clinical and Laboratory Standards Institute. Validation, Verification, and Quality Assurance of Automated Hematology Analyzers. 2nd ed. CLSI standard H26-A2. Clinical and Laboratory Standards Institute; 2010. Reaffirmed September 2016. Accessed September 26, 2026. https://clsi.org/shop/standards/h26/
  3. Clinical and Laboratory Standards Institute. Reference and Selected Procedures for the Quantitative Determination of Hemoglobin in Blood; Approved Standard. CLSI document H15-A3. Wayne, PA: CLSI; 2000.
  4. Gulati G, Uppal G, Gong J. Unreliable automated complete blood count results: causes, recognition, and resolution. Ann Lab Med. 2022;42(5):515-530. doi:10.3343/alm.2022.42.5.515
  5. Marin MJ, van Wijk XMR, Boothe PD, Harris NS, Winter WE. An introduction to the complete blood count for clinical chemists: red blood cells. J Appl Lab Med. 2024;9(5):1025-1039. doi:10.1093/jalm/jfae031

Watch one

Read each decision and its explanation.

A CBC shows RBC 1.95 × 10⁶/µL, MCV 148 fL, and MCHC 45.3 g/dL, with a right-shifted, widened red-cell histogram. Hemoglobin is 13.1 g/dL, close to yesterday's 13.3 g/dL. What do you do before this CBC is released?

  1. Review the histogram. It is shifted right and widened, unlike the usual single peak.

    A right-shifted, widened red-cell histogram signals that the red-cell indices need review.

  2. Compare the results. RBC is low, MCV and MCHC are both raised, and hemoglobin is close to yesterday's value.

    Noting which results changed and which did not narrows the cause.

  3. Identify the pattern. Red-cell autoagglutination, most often from cold agglutinins, falsely lowers the RBC count and falsely raises the MCV and MCHC.

    Clumped red cells cross the aperture as fewer, larger particles, which produces this pattern.

  4. Warm the specimen to 37 °C and rerun it immediately.

    Warming dissociates the agglutinates before they reach the aperture.

Release the red-cell results from the warm rerun. If the red cells clump again, replace the plasma with warm saline and repeat the count, so the agglutinates cannot reform before the cells reach the aperture.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A CBC reports an MCV of 134 fL and an MCHC of 42 g/dL with a low RBC count, and the red-cell histogram is right-shifted. What is the likely cause and the correction?

Incorrect. An MCHC above 40 g/dL is implausible for a true red-cell population and signals interference, so these indices are not released as measured.

Incorrect. Lipemia raises hemoglobin and MCH through turbidity. The low RBC count and right-shifted red-cell histogram come from clumped red cells.

Correct. Red-cell autoagglutination lowers the count and inflates MCV and MCHC, and the right-shifted histogram is the instrument clue. If agglutination returns after warming, a warm saline replacement is used.

Hint
  1. Compare the MCV and MCHC with values a true red-cell population can reach.
  2. A right-shifted histogram indicates cells or clumps larger than usual.
  3. Consider what warming the specimen to body temperature would reverse.

Review Spurious results and analyzer interference

Problem 2 of 3 · Hints available

A specimen with a markedly raised leukocyte count has a hemoglobin of 19.8 g/dL. Its hematocrit and RBC count are both close to yesterday's values. What do you check before this hemoglobin is released?

QC shows the analyzer measured its control material correctly. It cannot detect a specimen's own high leukocyte count adding turbidity to the photometric reading.

Reported a turbidity-raised hemoglobin without correction

Lipemia, very high leukocyte or platelet counts, unlysed cells, and precipitated paraproteins scatter light and falsely raise absorbance at 540 nm. The false hemoglobin then raises MCH and MCHC and breaks the rule of three (hemoglobin × 3 ≈ hematocrit). A plasma blank, further dilution, or another method-specific correction comes before the indices are released.

A markedly raised leukocyte count adds turbidity at the wavelength the photometric method reads, which can falsely raise the hemoglobin. The rule of three, applied to a hematocrit and RBC count that have not changed, detects the mismatch.

Those two results are close to the patient's baseline. A raised leukocyte count adds turbidity to the photometric hemoglobin measurement.

Hint
  1. Compare the hemoglobin with what the hematocrit and RBC count would predict.
  2. Consider what the extra leukocytes add to a photometric reading.

Review Quantitative hemoglobin: the cyanmethemoglobin method

Problem 3 of 3 · On your own

A patient with sickle cell anemia has a spun microhematocrit of 40% and an automated hematocrit of 33% on the same specimen. Which result is more likely biased, and by what?

Impedance counting and sizing works in sickle cell anemia. The known bias in this disease affects the spun method.

Plasma trapped between cells in sickle cell anemia biases the packed column upward, so a spun hematocrit can be higher than the automated result.

A discordance this size between the two methods has a known cause here. Investigate it with both methods' limitations in mind before either result is reported.

Assumed the packed column contains red cells only

Plasma trapped between cells in sickle cell anemia, spherocytosis, thalassemia, and hypochromic or macrocytic anemias biases the spun hematocrit upward, so it can exceed the analyzer value. A leaking seal or short draw lowers it, and under-centrifugation or reading the buffy coat raises it.

Review Microhematocrit

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Dexter Rose, an adult man (MRN 5038816), has a CBC drawn in the emergency department.
  • His CBC 3 days ago was unremarkable.
  • A chemistry panel drawn at the same time reports a glucose of 1,050 mg/dL.
  • The analyzer counts and sizes red cells by impedance after mixing them into an isotonic diluent.
TestResultPreviousReference intervalFlag
RBC4.62 × 10⁶/µL3 days ago: 4.65 × 10⁶/µL4.65 × 10⁶/µL3 days ago4.20–5.90 × 10⁶/µL4.20–5.90
Hemoglobin14.2 g/dL3 days ago: 14.4 g/dL14.4 g/dL3 days ago14.0–18.0 g/dL14.0–18.0
Hematocrit48.4 %3 days ago: 42.3 %42.3 %3 days ago42.0–50.0 %42.0–50.0
MCV104.8 fL High3 days ago: 91.0 fL91.0 fL3 days ago80.0–98.0 fL80.0–98.0High
MCH30.7 pg3 days ago: 31.0 pg31.0 pg3 days ago28.0–32.0 pg28.0–32.0
MCHC29.3 g/dL Low3 days ago: 34.3 g/dL34.3 g/dL3 days ago33.0–36.0 g/dL33.0–36.0Low

Specimen: EDTA whole blood.

Decision 1 of 3

Why is the MCV 104.8 fL today?

Clumped red cells lower the RBC count and raise the MCHC. Here the RBC count matches 3 days ago, and the MCHC is low.

Red cells in plasma with this much glucose contain extra intracellular glucose. In the isotonic diluent, water moves into the cells and they swell before they reach the aperture, so each pulse measures a larger cell.

Red cells live about 120 days, so the average cell size cannot rise this far in 3 days. The RBC count and hemoglobin have changed very little.

Review Automated counting principles

Decision 2 of 3

The hematocrit of 48.4% is within its reference interval. Is it reliable?

A result within the reference interval can be wrong. The rule of three predicts about 14.2 × 3 = 42.6%, and 48.4% does not fit.

This analyzer calculates the hematocrit from the RBC count and the MCV. The falsely high MCV falsely raises the hematocrit, and the MCHC calculated from that hematocrit is falsely low.

The RBC count of 4.62 × 10⁶/µL matches the count from 3 days ago. The error is in the cell size.

Review Microhematocrit

Decision 3 of 3

What do you do before the red-cell results are released?

A comment leaves the wrong MCV, hematocrit, and MCHC in the chart as the patient's values.

Mix a portion of the specimen with the analyzer's diluent and let it stand at room temperature for about 10 minutes. The cells finish swelling before the count, and the rerun results are corrected for the dilution. A spun microhematocrit is another check, because glucose does not affect it.

The cells swell in the diluent on every run, so an immediate rerun repeats the same error.

Review Automated counting principles

The high MCV with a low MCHC points to red cells swelling in the diluent:

  • The MCV rose 13.8 fL in 3 days, and the RBC count and hemoglobin changed very little.
  • The MCHC fell below its reference interval.
  • The glucose of 1,050 mg/dL is well above the 600 mg/dL level where this swelling can begin.4

Most analyzers calculate the hematocrit from the MCV and the RBC count, so the hematocrit and the MCHC have the same error.5

For reference

Sources checked