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How analyzers count cells and measure hemoglobin

13 min

  • 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

Read the full reference

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?

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

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.

When two cells cross together

Two cells crossing the aperture at the same moment generate a single, oversized pulse. The count reads low, and the volume histogram reads high, because the instrument has counted one large event in place of two ordinary ones. Red-cell autoagglutination, most often from cold agglutinins, produces this same pattern across a whole specimen: fewer, larger apparent cells, a red-cell count that reads low, and an MCV and MCHC that read high. Warming the specimen to 37 °C and rerunning it promptly reverses the agglutination for the count that follows.4

Interference the count does not share

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 built from it can stay close to their own baseline. Checking the rule of three, hematocrit near 3 times the hemoglobin, catches this kind of mismatch before a result is released.4

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

Watch one

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. Read the histogram: it is shifted right and widened, a different shape from its usual single peak.

    A right-shifted, widened red-cell histogram is the instrument's own clue to look past the numbers.

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

    Checking which results moved and which did not narrows the cause quickly.

  3. Recognize the pattern: red-cell autoagglutination, most often from cold agglutinins, undercounts the cells and inflates their apparent volume and concentration.

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

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

    Warming dissociates the agglutinates before they reach the aperture.

  5. If the red cells reagglutinate on the rerun, replace the plasma with warm saline and repeat the count.

    Agglutination that returns needs the red cells in warm, cell-free plasma to keep it from reforming before the next count.

Warm the specimen to 37 °C and rerun the CBC immediately. A warm saline replacement is used if the agglutination returns.

Your turn

Problem 1 of 3

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 describes cells or clumps larger than usual.
  3. Think about what warming the specimen to body temperature would undo.

Review Spurious results and analyzer interference

Problem 2 of 3

A specimen with a markedly raised leukocyte count reads a hemoglobin of 19.8 g/dL. Its hematocrit and RBC count are both close to yesterday's ordinary 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, against a hematocrit and RBC count that have not moved, catches the mismatch.

Those two results are close to their own baseline. The result a raised leukocyte count adds turbidity to is the photometric hemoglobin.

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

Review Quantitative hemoglobin: the cyanmethemoglobin method

Problem 3 of 3

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 runs the other way, in the spun method.

Plasma trapped between cells in sickle cell anemia biases the packed column upward, so a spun hematocrit can read 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

  • A CBC on an adult woman (MRN 6620475) is drawn on a medical ward.
  • Her CBC yesterday was unremarkable: hemoglobin 12.8 g/dL, hematocrit 38%, RBC count 4.70 × 10⁶/µL.
  • The collection note for today's specimen says the plasma looked visibly lipemic, and a fasting lipid panel is pending.
  • No transfusion order appears in her chart between the two draws.
TestResultPreviousReference intervalFlag
RBC4.68 × 10⁶/µL4.70 × 10⁶/µLYesterday4.20–5.90 × 10⁶/µL
Hemoglobin17.9 g/dL12.8 g/dLYesterday12.0–16.0 g/dLHigh
Hematocrit39 %38 %Yesterday37.0–47.0 %
MCV83.3 fL80.9 fLYesterday80.0–98.0 fL
MCH38.2 pg27.2 pgYesterday28.0–32.0 pgHigh
MCHC45.9 g/dL33.7 g/dLYesterday33.0–36.0 g/dLHigh

Specimen: H 4, L 88, I 1. EDTA whole blood; plasma visibly lipemic at collection.

Decision 1 of 3

What does this pattern point to?

Hemoglobin does not rise this much in a healthy marrow over one day. The RBC count and hematocrit, which would rise with a true increase, have barely moved.

The lipemia index is far above its usual range. Turbidity from the lipemia raises the photometric hemoglobin reading, and MCH and MCHC rise with it because both are calculated from that hemoglobin.

A dilution error affecting the whole count would move the RBC count and hematocrit as well. Both sit close to yesterday's values.

Review Automated counting principles

Decision 2 of 3

What do you do with the hemoglobin before it is released?

A comment does not correct the number. The turbidity-raised hemoglobin, MCH, and MCHC would still reach the chart as the patient's own values.

A plasma blank, further dilution, or a platform's own back-calculated hemoglobin removes the turbidity's contribution before the corrected value and its indices are released.

A second run measures the same turbid plasma and reads the same falsely raised value. Agreement between two runs of the same tube does not correct the interference.

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.

Review Quantitative hemoglobin: the cyanmethemoglobin method

Decision 3 of 3

Does the hematocrit need the same correction as the hemoglobin?

Hematocrit here sits close to yesterday's value, at 39% against 38%. Being reported on the same panel does not mean it shares the hemoglobin's interference.

Turbidity raises absorbance at the wavelength the photometric hemoglobin method reads. The RBC count and MCV, and the hematocrit built from them, are measured a different way and stay close to their own baseline.

Every specimen on this analyzer is diluted for the count, including one without lipemia. Dilution is not what separates the affected result here from the unaffected ones.

Review Microhematocrit

The clue that settles this case is what moved and what did not:

  • Hemoglobin, MCH, and MCHC all rose sharply in a day with no transfusion to explain it.
  • RBC, hematocrit, and MCV stayed close to yesterday's values.
  • The lipemia index is far above its usual range, and the specimen was visibly lipemic at collection.

Turbidity from the lipemia reaches the photometric hemoglobin reading and the indices calculated from it. It does not reach the red-cell count, the hematocrit, or the MCV.

Keep

Sources checked