American Society for Veterinary Clinical Pathology
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Madison, WI 53704

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2.4. Manual Hematology for Non-mammalian Species


2.4.1. Monitoring.

Internal monitoring should include the following: monitor reagent preparation for cell counting diluent (reagent grade water, verification of quality of new lot as compared with previous)

2.4.2. Method Validation – See also General recommendations.

Analysts must be proficient in exotic animal hematology but will otherwise perform standard method validation tests. Not all of the method validation experiments listed in section 2.1.2. may be applicable to evaluation of exotic manual hematology methods.  Method validation experiments should be selected or modified as necessary to ensure that new methods are functioning satisfactorily to meet the laboratory's requirements and the manufacturer's specifications.

2.4.3. Instrumentation – see also General recommendations.

Equipment (e.g. hemacytometers, weighted hemacytometer cover slips, hand tallies, calibrated pipettes, differential cell counters) used for hematology procedures should be in good working order. Routine monitoring and regular maintenance of each piece of equipment (e.g. annual calibration of pipettes and balances) should be performed and documented. Records of maintenance, and malfunction and repairs should be kept.   Abbott Diagnostics, Inc. (Abbott Park, Illinois) has validated and supports automated cell counts on some non-mammalian species using the automated hematology analyzers, the Cell-Dyne 3500 or higher.  Validation has been done at Sea World Laboratories.

2.4.4. Personnel Knowledge.

Laboratory analysts must be proficient in cell identification for the species tests.  Comprehensive knowledge of species variation when using flow cytometry is important to understand when verification by manual methods is required.

2.4.5. Quality Control.

Manual WBC counts using a hemacytometer are imprecise and have coefficients of variation ranging from 20-40% (Schalm, Harr et al, 2005); therefore, quality control implementation and statistical analysis may yield significance or a lack of significance that is not relevant to daily operation.  Method validation studies for shark species conducted by J. Arnold (2005) showed coefficients of variation comparable to manual hematology for human WBC counts as reported in the B-D product insert for Unopette 365855 when the sample was processed within 5 hours of collection.

a. Reagents and Materials.  Documented protocols for cell counts include direct count using Methyl Violet (Natt & Herrick, 1952) to determine total RBC, WBC and thrombocyte counts, direct count with no dye (Hawkey, 1988) or an indirect method using Phloxine B dye (Campbell, 1988) for total WBC only.  In the latter method, only the cells containing eosinophilic granules are stained and the total WBC is calculated based on the percent heterophils and eosinophils from the differential. 


Reported method validation studies between the direct and indirect techniques are conflicting (Dein, 1994; Arnold, 1995) and require further investigation, preferably following Westgard's guidelines for Method Validation, with a more representative number of animal species.  The disparity in results may be due to the imprecision of each method.  The diluent described by Natt and Herrick can be prepared in the laboratory and it is suitable for all non-mammalian vertebrate species, however, when this diluent is used for some chelonian (turtle) and elasmobranch species (sharks, skates and rays), additional salts are required to adjust the osmolality of the stock solution (Arnold, 2005).  Hawkey's technique utilized Becton Dickinson's WBC Unopette. Campbell's method using Phloxine B diluent is no longer available as Eosinophil Unopette 5877, but a comparable diluent can be prepared by the user.


b. Currently, commercially prepared control materials are not available for non-mammalian blood cell counts.  Procedural controls include:

i. Duplicate dilutions from a patient specimen, performed within the acceptable time limits for sample stability.

ii. WBC Estimate from Blood Smear.  Each institution should document a protocol to achieve a reliable method for evaluating the accuracy of the hemacytometer counts.  Estimated total WBC values may be difficult due to the similar morphology of lymphocytes and thrombocytes when viewed at lower magnifications typically used for WBC estimates of mammalian cells.


c. Proficiency testing for technologists should be documented annually, or more frequently as determined by the institution.  Testing should include comparison counts from the same blood sample for total cell counts and for leukocyte differentials.  Sample selection should be representative of patient population (avian, reptile, teleost, elasmobranch, etc.).  Between technologists, hemacytometer counts should agree to within 15% and differential percentage results for each cell type should agree to within 95% confidence interval.


Direct Cell Count Method—Thrombocyte/Lymphocyte Error.  It may be difficult to differentiate between thrombocytes and lymphocytes in the hemacytometer for newly trained technologists, or for experienced technologists when counting some animal species.  A good quality control check is to count all of the non-erythrocytes in the nine large squares of the chamber and calculate the total number (this is not the actual value and must be corrected from the differential).  Perform the differential twice - include the thrombocytes in the first differential and exclude them in the second.  The latter is reported as the actual differential results.  Using the percent thrombocytes from the first differential, calculate the absolute value and then subtract it from the total number to determine the total WBC.  Example: Tally from hemacytometer with a 1:100 dilution = 750 non-erythrocytes cells;  750 × 1.1 × 100 = 82,500/µL for the total non-erythrocytes count.  The first differential = Monocytes (1%), Lymphocytes (9%), Heterophils (8%) and Thrombocytes (82%) so the absolute value of the thrombocytes = 0.82 × 82,500 = 67,650.  The total WBC = 82,500 – 67,650 = 14,850/µL.

2.4.6. Procedures Manual – see General recommendations

2.4.7. Comparison of Tests – see General recommendations

2.4.8. Identification of out-sourced tests – see General recommendations