Choosing the Right Leukocyte Counting Method in Non-Mammalian Vertebrates

For veterinarians and animal scientists working with birds, reptiles, amphibians, and/or fish, obtaining accurate hematological data is vital for diagnosing disease, monitoring immune response, and assessing overall health. A key part of this process is determining leukocyte (white blood cell) counts. Two commonly used techniques for these species are Phloxine B and Natt & Herrick’s Solution (NHS) (Natt & Herrick, 1952). Each has their own strengths and limitations.

Semidirect Method: Phloxine B

Phloxine B is widely appreciated for its ease of use and precision (Gaunt, 2004). When used with a Neubauer ruled hemacytometer, it often yields more consistent WBC counts than Natt & Herrick technique (Dein et al., 1994). This makes it a practical choice for routine hematological evaluation in clinical or research settings.

However, this method does not measure the total WBCs directly. Phloxine B stains granulocytes, including heterophils, which are the avian equivalent to the mammalian neutrophil, and eosinophils, which function in immunity like their mammalian counterpart (Figure 1). This becomes a drawback when mononuclear leukocytes outnumber granulocytes, as it can skew results and reduce the accuracy of the total counts. This limitation should be kept in mind, particularly in species where mononuclear dominance is common (Campbell & Ellis, 2013).

Direct Method: Natt and Herrick’s

Where more direct quantification of total leukocytes is necessary, especially when cell population is unpredictable, the Natt and Herrick’s method provides a more reliable approach. This technique involves diluting a blood sample at a 1:200 ratio with Natt and Herrick’s solution, which stains the leukocytes dark blue and allows for straightforward identification (Campbell & Ellis, 2013).

While this method may be slightly more time-consuming, the advantage of using this method is that a total erythrocyte (red blood cell) and thrombocyte count can also be obtained (Campbell & Ellis, 2013).

Staining Protocol

Phloxine B Staining Procedure

1. 1:20 Dilution: Mix 20 µL of whole blood with 380 µL of Phloxine B solution.
2. Incubate: Let the solution mixture stand in a humid chamber for 3-5 minutes.
3. Loading: Gently load the stained mixture into a Neubauer-ruled hemacytometer.
   • Note: Prolonged exposure will falsely stain RBCs.
4. Counting: Perform routine TWBC and total heterophil + eosinophil counts.

The following are suggested procedures for the Natt-Herricks technique (NHT) (Campbell & Ellis, 2013; Fudge, 2000):

Natt and Herrick’s Staining Procedure

1. Dilution: Mix 10 µL of whole blood with 1990 µL of Natt and Herrick’s Solution
2. Mix: Invert or gently shake to ensure even cell distribution without lysing cells.
3. Incubate: Let mixture stand for 10-15 minutes at room temperature.
4. Loading: Charge a clean Neubauer-ruled hemacytometer chamber with the stained sample.
5. Counting: Perform routine counting methods for TRBC and TWBC in duplicate with 15% agreement between the two sides.

Note: If there is difficulty differentiating between lymphocytes and thrombocytes, allow the sample to incubate in the Natt and Herrick’s for 60 minutes. This improves the differentiation between small lymphocytes and thrombocytes.

Natt and Herrick’s Staining Procedure with Cell Diluting Pipettes

1. Dilution: Using a cell-diluting pipette, draw whole blood to the 0.5 mark.
2. Mix: Draw the Natt and Herrick’s solution to the 101 mark.
3. Incubate & Load: Load into a Neubauer-ruled hemacytometer and allow to settle for a minimum of 5 minutes.
4. Counting: Perform routine counting methods for TRBC and TWBC in duplicate with 15% agreement between the two sides.

Citations

Campbell, TW, & Ellis, CK. (2013). Avian and exotic animal hematology and cytology. John Wiley & Sons.

Dein, FJ, Wilson, A, Fischer, D, et al. (1994). Avian leucocyte counting using the hemocytometer. Journal of Zoo and Wildlife Medicine, 432-437.

Fudge, AM. (2000). Avian complete blood count. Laboratory Medicine: Avian and Exotic Pets. Philadelphia, PA: WB Saunders, 16, 9-18.

Gaunt, SD. (2004). 9 – laboratory evaluation of leukocytes. In RL Cowell (Ed.), Veterinary clinical pathology secrets (pp. 33-37). Mosby. https://doi.org/https://doi.org/10.1016/B978-1-56053-633-8.50011-5

Natt, MP, & Herrick, CA. (1952). A new blood diluent for counting the erythrocytes and leucocytes of the chicken*. Poultry Science, 31(4), 735-738. https://doi.org/https://doi.org/10.3382/ps.0310735

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