Lecture 2: Hematology instruments & blood cell measurements

Which erythrocyte parameters are routinely reported on a CBC?

HCT, HGB, RBC count, plus erythrocyte indices (MCV, MCH, MCHC, and sometimes RDW).

What leukocyte parameters are routinely reported on a CBC?

Total leukocyte count and a differential leukocyte count.

What platelet parameters may appear on a CBC?

Platelet count and sometimes MPV.

What do impedance analyzers measure directly, and how is HGB obtained?

They count and size cells (cell volume distributions). HGB is measured spectrophotometrically.

Which CBC outputs are typically reported by impedance instruments?

HCT, RBC, HGB, MCV, MCH, MCHC, RDW, total WBC (all species), and generally platelet counts.

Key limitations of impedance technology for cell characterization?

Cannot determine intracellular characteristics; provides only a 3-part differential based on size—limited accuracy vs. 5-part.

Species caveat for impedance platelet counts?

Cats—impedance cannot do platelet counts accurately.

Precision and cost profile of impedance analyzers?

Excellent precision and generally less expensive than laser flow instruments.

Can impedance analyzers perform reticulocyte counts?

No, they cannot perform reticulocyte counts.

Fundamental signal principles behind laser flow cytometry in hematology?

Light scatter: cells crossing a laser produce forward scatter (size) and side scatter (internal complexity); classification uses extinction of light and scattered light.

What does forward scatter primarily reflect, and give examples?

Cell size: larger cells (e.g., neutrophils) → larger forward scatter; smaller (lymphocytes) → smaller.

What does side (90°) scatter reflect, with examples?

Internal complexity/granularity: neutrophils (multilobed, granular) → high side scatter; lymphocytes (scant cytoplasm) → lower side scatter.

Core capabilities of laser flow analyzers for cell assessment?

Determine number, size, and internal complexity; count and classify via light extinction + scatter.

Which CBC/indices are reported by laser flow systems?

HCT, RBC, HGB, MCV, MCH, MCHC, RDW.

What WBC outputs do laser flow cytometers provide, and an important canine exception?

Total WBC and 5-part differential; basophils are not accurately counted in dogs.

Platelet metrics available on laser flow cytometers?

Platelet count, MPV, PDW, PCT.

Do laser flow cytometers provide reticulocyte data?

Yes — reticulocyte counts are available.

Example of a high-end laser flow instrument and its implication?

Large platforms (e.g., Advia 2120) offer many additional measurements beyond core parameters.

Quick compare: Impedance vs. Laser — differential capability

Impedance: 3-part (size-based, limited). Laser: 5-part (scatter-based), except canine basophils not reliable.

Quick compare: Reticulocytes & intracellular detail

Impedance: No reticulocytes, no intracellular detail. Laser: Reticulocytes + intracellular complexity via side scatter.

Quick compare: Cost/precision snapshot

Impedance: Less expensive, excellent precision. Laser: More expensive, richer data (size + complexity).

What does MCV represent, what units are used, and how is it determined?

Mean Cell Volume (MCV): average erythrocyte volume (in femtoliters, fL = 10⁻¹⁵ L). Determined directly by hematology analyzers.

What does MCH represent, and why is it considered less clinically useful?

Mean Cell Hemoglobin (MCH): average Hb content per RBC (in picograms, pg = 10⁻¹² g). Correlates with MCV and partly internal Hb concentration. Adds little beyond MCV since both track together.

In what condition is MCH expected to reach its lowest values?

Severe iron deficiency anemia (due to low MCV and low Hb concentration per RBC).

How is MCHC calculated, and how does its denominator differ from whole blood Hb?

Mean Cell Hemoglobin Concentration (MCHC):

(Hb÷HCT)×100

Reported in g/dL of packed erythrocytes, not whole blood.

Example: Hb = 15 g/dL, HCT = 45% → (15/45) × 100 = 33 g/dL.

Why is MCHC more diagnostically useful than MCH?

Unlike MCH, MCHC reflects relative Hb concentration within RBCs and can identify true hypochromasia or artifact (hemolysis, lipemia) when spuriously increased.

What does RDW represent, and how is it calculated?

Red Cell Distribution Width (RDW): coefficient of variation of erythrocyte volumes.

(SDofRBCvolumes÷MCV)×100

Provides an electronic measure of anisocytosis.

What are common causes of spuriously increased RDW?

Erythrocyte agglutination

Platelets miscounted in the RBC histogram (especially in severe anemia).

Which index directly measures cell size, which directly measures Hb content, and which is a calculated concentration?

MCV: Direct measure of cell size.

MCH: Direct measure of Hb content per cell.

MCHC: Calculated concentration of Hb in RBC mass.

Which RBC index best quantifies anisocytosis electronically?

RDW, since it reflects variability in RBC volumes relative to MCV.

Which indices may be falsely altered by hemolysis or analyzer artifacts?

MCHC: spuriously ↑ with hemolysis or lipemia.

RDW: spuriously ↑ with agglutination or platelet interference.

What technology do most automated analyzers use to count reticulocytes?

Flow cytometry, which classifies cells based on light scatter/extinction (platform dependent).

What quality assurance step is required for absolute reticulocyte counts from flow cytometry?

Validate automated absolute reticulocyte counts with manual counts.

In feline samples, which reticulocyte class is typically counted by most instruments?

Only aggregate reticulocytes (most instruments exclude punctate forms).

Why does it matter that many analyzers count only feline aggregate reticulocytes?

It affects interpretation of regeneration—aggregate retics reflect current regeneration, while punctate retics persist longer and may be undercounted.

List two pre-analytical/analytical issues that can spuriously increase RDW.

Erythrocyte agglutination and platelets miscounted in the RBC histogram (especially in severe anemia).

Define "spurious reticulocytosis."

Falsely elevated reticulocyte counts due to non-reticulocyte elements that the analyzer flags as retics.

Name two infectious causes of spurious reticulocytosis.

Hemotropic mycoplasmas and Babesia spp. (contain RNA/DNA).

Name two erythrocyte inclusions that can cause spurious reticulocytosis.

Howell-Jolly bodies (micronuclei) and nucleated erythrocytes.

Which platelet-related artifacts can mimic reticulocytes on automation?

Large immature platelets and platelet clumps.

Which oxidative inclusion can nonspecifically fluoresce and elevate automated retic counts?

Heinz bodies (nonspecific fluorescence).

Which non-cellular factors can cause autofluorescence and spuriously raise retic counts?

Drugs and porphyrin (autofluorescence).

Can leukocytosis cause spurious reticulocytosis?

Yes, but uncommonly.

Practical step when automated retic count seems discordant with smear review?

Perform/confirm a manual reticulocyte count and review a blood smear for artifacts (agglutination, large platelets, inclusions).

Why is manual validation critical in anemic patients with severe thrombocytosis or platelet clumping?

Platelet aggregates/giant platelets can be misclassified as retics, falsely elevating the automated retic count.

Quick check: two common causes of spuriously increased RDW you should rule out before over-interpreting anisocytosis?

Agglutination and platelet interference in the RBC histogram.

Why is it essential to always review a stained blood film in addition to automated counts?

Because artifacts, aggregates, and abnormal cells can lead to spurious results on analyzers that can only be recognized by microscopic confirmation.

What is the most common cause of false thrombocytopenia on automated counters?

Platelet aggregates, often due to platelet activation during blood collection/handling (especially in cats).

Which hematology method is especially unreliable for feline platelet counts?

Impedance counters (automated platelet counts often inaccurate in cats).

Name two causes of spuriously increased mean platelet volume (MPV).

Platelet aggregates and storage of blood at 5 °C.

How do nucleated erythrocytes (NRBCs) interfere with leukocyte counts?

NRBCs are misclassified as WBCs by manual and most automated counters, falsely elevating the leukocyte count.

What formula is used to calculate a corrected WBC count when NRBCs are present?

Corrected WBC = Machine WBC × 100 ÷ (100 + NRBC per 100 WBC).

A patient's analyzer WBC = 15 × 10³/µL, and 50 NRBC/100 WBC are seen on smear. What is the corrected WBC?

10 × 10³/µL (15 × 100 ÷ 150).

What artifact can old blood samples cause regarding RBC indices?

Erythrocyte swelling, leading to ↑MCV, ↑HCT, and altered MCHC.

How do old samples affect platelet measurements?

Platelet aggregation, resulting in ↓platelet count and ↑MPV.

How does cell lysis in old samples affect automated results?

Counts of all cell types are decreased.

How can aggregates of RBCs, platelets, or WBCs skew analyzer data?

↓measured counts per µL

RBC aggregates → ↑MCV, ↑HCT, abnormal MCHC

Platelet aggregates → miscounted as WBCs, ↑MPV

How can anticoagulant choice reduce aggregates in samples?

Citrate anticoagulant may reduce platelet and leukocyte aggregation.

What happens to automated counts in the presence of clot formation?

All cell types appear decreased.

What are cryoglobulins, and how do they interfere with results?

Proteins that precipitate below body temp; may be misclassified as WBCs or platelets.

How does hemolysis affect MCHC?

Causes a spuriously increased MCHC, from either in vivo (intravascular hemolysis) or in vitro (sample handling).

Which two sample abnormalities can falsely elevate hemoglobin and MCHC?

Lipemia and Heinz bodies.

How does lipemia affect analyzer measurements beyond Hb and MCHC?

May also spuriously increase leukocyte and platelet counts.

How do Heinz bodies affect reticulocyte counts?

Their nonspecific fluorescence can increase reticulocyte counts on flow cytometry.

Why should dot plots and histograms from analyzers always be examined?

They help detect aggregates, abnormal cell populations, or artifacts that may not be obvious from numeric values alone.

Why does the RBC count alone have little diagnostic value?

Because it is mainly used to calculate hematocrit (Hct) electronically and to derive MCV and MCH manually.

What are the two methods of determining hematocrit (Hct/PCV)?

Centrifugation → packed cell volume (PCV, "gold standard").

Automated analyzers → calculated Hct from MCV and RBC count.

What formula do automated analyzers use to calculate hematocrit (Hct)?

Hct (%) = (MCV × RBC) ÷ 10.

Example: If MCV = 68 fL and RBC = 6.6 × 10⁶/µL, what is the Hct?

45% (68 × 6.6 ÷ 10).

How is hemoglobin concentration determined in hematology analyzers?

By a spectrophotometric assay using sodium lauryl sulfate (SLS).

What does sodium lauryl sulfate (SLS) do in hemoglobin determination?

Lyses RBCs

Oxidizes Hb to MetHb

Forms a stable SLS-MetHb complex for spectrophotometric measurement.

Q7. What conditions cause falsely increased hemoglobin values?

Lipemia

Heinz bodies

Nuclei in non-mammals (since nucleated RBCs add absorbance).

Why can PCV and Hct differ in accuracy?

PCV (centrifugation) directly measures packed RBCs, while Hct is calculated, and therefore more susceptible to analyzer errors (e.g., spurious RBC count or MCV).