Methodological Considerations for Using Natt & Herrick Solution in Hematological Studies

Introduction

The Natt & Herrick solution is a diluent‐stain mixture used in manual hematology, particularly for species with nucleated erythrocytes (such as birds, reptiles, fish). It lyses or at least renders erythrocytes less interfering by staining them (with their nuclei), while also staining leukocyte nuclei, allowing total leukocyte (WBC) and erythrocyte (RBC) counts using a hemocytometer. Because of its manual nature, a number of methodological variables can affect accuracy and reproducibility. It is essential for labs to adopt standard best practices to reduce variability in leukocyte counting across labs.

The Biochemical Principle: How Natt & Herrick Solution Works

• The solution is buffered saline containing a dye (commonly methyl violet 2B), formalin (to fix cellular components), salts (NaCl, Na₂SO₄, phosphate buffers), etc. productosveterinarioseurovet.com+2Wiley Online Library+2

• Erythrocyte (nucleated in many non‐mammalian species) nuclei remain visible/stained, but the cytoplasm is less distinct; this helps avoid miscounting in hemocytometer grids. Leukocytes are stained more intensely (blue to purple) via the nuclear stain, allowing discrimination. PMC+2Wiley Online Library+2

• Some components also act partially to “soften” or lyse the cytoplasmic membranes of erythrocytes, reducing background, but the solution generally does not completely lyse nucleated erythrocytes in species where they are large and robust. The dye plus fixative helps preserve morphology so that leukocytes can be distinguished. Variability in staining intensity depends on freshness, composition, and how long the stain has aged.

Sample Preparation: Best Practices

 Blood Collection & Anticoagulant Choice

• Anticoagulant: EDTA (especially K₂ or K₃ EDTA) is commonly used for avian blood and many other non-mammalian vertebrates. Heparin is sometimes used, but EDTA is preferred because it better preserves leukocyte morphology. Some anticoagulants may cause hemolysis (or partial erythrolysis) over time. productosveterinarioseurovet.com+2ijvm.org.il+2

• Volume and quality: Use sufficient volume to allow for repeats or QC. Avoid hemolysed or heavily clotted samples. Draw carefully to avoid trauma; avoid squeezing or otherwise damaging the cellular elements.

 Freshness and Storage

• Use the sample as soon as possible after collection. Delay leads to morphological changes (e.g. leukocyte swelling, fragmentation), erythrocyte degeneration, hemolysis, and changes in viability of leukocytes. ijvm.org.il+2productosveterinarioseurovet.com+2

• If storage is necessary: Keep at cool temperatures (4 ‒ 8 °C) for short durations. Prolonged storage (over several hours) leads to changes. Some studies show EDTA exposure beyond certain hours induces erythrolysis or cell morphology change in non‐mammalian species. ijvm.org.il+2USGS+2

 Dilution Ratios

• The canonical dilution with Natt & Herrick solution is 1:200 (one part whole blood to 199 parts diluent) for many avian or non‐mammalian blood cell counts. productosveterinarioseurovet.com+2USGS+2

• Some variant protocols might use slightly different dilutions if counting smaller or larger cells, or if the hemocytometer grid area used changes. But changing the dilution must be validated and consistently applied.

• Ensure accurate volumetric techniques: When using capillary tubes, pipettes, or calibrated diluting pipettes, avoid carryover, bubble formation, etc. Pre‐wet pipette tips/diluting containers to avoid loss by adhesion.

 Microscopy / Hemocytometer Setup & Counting Protocols

 Hemocytometer Type & Grid Selection

• Use a Neubauer improved counting chamber or equivalent (with standardized grid dimensions). The improved Neubauer has ruled large squares (e.g. nine large squares for WBC) and smaller sub‐squares. productosveterinarioseurovet.com+2USGS+2

• Clean thoroughly before use. Ensure coverslip is clean and properly seated to give correct chamber depth. Avoid dirt, dust, or manufacturing defects.

 Microscope Magnification & Illumination

• Typically, use 40× objective (i.e. 400× total magnification) for erythrocyte/RBC counting (to see stained nuclei) and possibly for leukocytes if size permits. For WBC counts, depending on leukocyte size and staining contrast, lower objectives (10×) may be used with caution. But many protocols recommend 40× for both to avoid missing small leukocytes. productosveterinarioseurovet.com+2PMC+2

• Illumination: Bright‐field illumination is standard. Phase contrast may help in some species or if background is problematic. The condenser should be adjusted so that there is even illumination, and that the contrast is sufficient to see nuclei distinctly.

 Loading the Chamber, Settling Time, and Counting Rules

• Mix the diluted sample thoroughly but gently (avoid foaming).

• Load the hemocytometer by touching capillary tip to the edge of the coverslip carefully; avoid bubbles. Do not overload or underfill the chamber.

• Allow settling time (often ~3 minutes) so that cells can settle into the focal plane and avoid drift. Some protocols specify incubation at room temperature during this. productosveterinarioseurovet.com+1

• Counting squares:

  • For RBC (or nucleated cells): Count in specified squares (e.g. four corner + central square of the central large square of the hemocytometer grid) to get RBC count. Scribd+2productosveterinarioseurovet.com+2

  • For WBC: Count all leukocytes in the nine large ruled squares (for standard Neubauer) or equivalent. Count cells touching left and top boundary lines; do not count those touching right or bottom borders to avoid double counting. Scribd+2productosveterinarioseurovet.com+2

Calculations

• Formula:

  Cells/µL=(Number of cells counted)×dilution factorvolume of counted squares (µL)\text{Cells/µL} = \frac{\text{(Number of cells counted)} \times \text{dilution factor}}{\text{volume of counted squares (µL)}}Cells/µL=volume of counted squares (µL)(Number of cells counted)×dilution factor​

• For example, with 1:200 dilution, if nine large squares total cover 0.9 mm² × chamber depth, etc., the conversion factor is standard. Many product inserts (e.g. Natt‐Herricks‐TIC 1:200) give explicit formulas. productosveterinarioseurovet.com+1

Sources of Variability & How to Control Them

Standardization Across Laboratories: Suggested Protocol Elements

To ensure that leukocyte counts using Natt & Herrick are reproducible across labs, here is a suggested standard protocol (template) with critical elements that labs should include in their SOPs / validation:

1. Sample collection

   • Anticoagulant: K₂‐EDTA or K₃‐EDTA.

   • Volume: sufficient for repeats.

   • Time of collection recorded.

2. Handling and transport

   • Transport at 4-8 °C when delay expected.

   • Process within a maximum specified time (e.g. 4 hours) unless validated otherwise.

3. Stain/Diluent handling

   • Use certified Natt & Herrick solution (or if lab‐made, follow validated recipe).

   • Filter before use if precipitates present.

   • Record batch number / date prepared.

   • Store in dark, cool environment.

4. Dilution

   • Dilution ratio: 1:200 (blood : Natt & Herrick).

   • Use calibrated pipettes or diluting capillaries.

   • Mix gently, avoid bubbles.

5. Hemocytometer preparation

   • Clean, dry counting chamber.

   • Coverslip properly seated.

   • Chamber depth verified (if possible).

6. Loading & settling

   • Load sample avoiding bubbles.

   • Wait ~3 minutes for settling.

   • Room temperature controlled (e.g. 18-25°C).

7. Microscopy

   • Use 40× objective for counting RBC and WBC unless otherwise validated.

   • Illumination standardized (e.g., bright‐field, condenser settings).

   • Use consistent counting squares (e.g. nine large squares for WBC).

   • Follow left/top border inclusion, exclude bottom/right borders rules.

8. Counting and calculation

   • At least two independent counts per sample (or duplicate).

   • Calculate cell concentration using dilution and volume of counted area.

   • Include formula in SOP.

9. Quality control and proficiency

   • Use control samples (known leukocyte counts) to check accuracy.

   • Inter‐operator comparison.

   • Document reproducibility (coefficient of variation) for repeat measurements.

10. Reporting

    • Report both raw counts (cells/µL or cells/mm³) and any corrections made (e.g. for dilution).

    • Note anticoagulant used, time since collection, temperature of storage.

    • If comparing with reference intervals, ensure those intervals were derived using same method.

Empirical Findings: What Studies Show

• In avian blood, studies comparing Natt & Herrick (direct hemocytometer count) vs smear‐based leukocyte estimation show that while there may be no constant or proportional bias in many cases, the limits of agreement can be wide, indicating random error. Hence, using the same method (Natt & Herrick) consistently in longitudinal studies is important. BioOne+2PMC+2

• Precision (repeatability) is often moderate: e.g. coefficient of variation (CV) of ~16–23% in some manual methods. PubMed

• In protocols like Natt‐Herricks‐TIC® (a commercial kit), performance in term of staining quality and countability of granulocytes was good; however operator training and non‐statistical quality control still required. PubMed+1

Special Considerations: Species, Erythrocyte Nucleation, and Alternative Methods

• In species with nucleated RBC’s (all birds, many reptiles, fish), the Natt & Herrick method is especially useful because automated counters typically fail (misclassify or cannot lyse properly). ScienceDirect+1

• In small mammals or where erythrocytes are non‐nucleated, simpler methods (standard RBC/WBC counting with different lysing reagents) may be preferable. But if using Natt & Herrick in such cases, validation is essential.

Recommendations for Validation and Inter‐Lab Harmonization

• Establish reference intervals for each species using the chosen method (blood collection, anticoagulant, time, dilution, counting rules). Many labs or academic veterinary medicine groups publish such intervals. BioOne

• Perform method agreement studies when new kits (e.g. Natt-Herricks-TIC) are adopted or when making significant protocol changes. Use Bland‐Altman plots, Passing‐Bablok regression etc. PubMed+1

• Include proficiency testing across operators within a lab and, if possible, across labs (interlab). Use standard specimens.

• Harmonize reporting: lab reports should always include method used (dilution, anticoagulant, counting chamber type, magnification).

Example Standard Protocol Summary

Below is a condensed version that could serve as a protocol summary in an SOP.

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Standard Operating Protocol: WBC Counting using Natt & Herrick

Conclusion

Using Natt & Herrick solution for leukocyte (and erythrocyte) counting remains valuable in many settings (veterinary, comparative physiology, species lacking automated hematology options). However, its manual nature means that variability can be high unless protocols are rigorously standardized. Labs wishing to ensure reproducibility should sharply define all steps (anticoagulant, sample age, dilution, counting rules, microscopy settings), validate their own reference intervals, and apply regular quality control.

Further Reading and Protocol References (edu / gov)

Here are several .edu / .gov resources to consult for detailed methods, validation studies, and reference intervals:

• “Avian Leucocyte Counting Using the Hemocytometer” by Dein, Wilson, Fischer, Langenberg. USGS (gov) — details comparing Natt & Herrick and Unopette methods. Publications de l’USGS

• “Hematologic methods in fish – Not only for beginners” (ScienceDirect, often containing methods from academic institutions) for application in fish species. ScienceDirect

• Studies from universities establishing reference intervals (e.g. cockatiels, etc.) via Natt & Herrick vs smear‐based estimation. BioOne