Comparison of Manual Total Cell Count with Fresh versus Formalin Fixed Blood from Fish
Jill Ellen Arnold, MS, MT(ASCP)
National Aquarium, 501 E. Pratt St., Baltimore, MD USA
jarnold@aqua.org
Total cell counts in fish species are a valuable tool for health assessment and monitoring,
however it can be challenging to process the specimens before cell distortion and deterioration
affects analytical precision. This occurs by 5-7 hours in EDTA in the sandbar shark,
Carcharhinus plumbeus 1. Research in crustacean hemocyte counts recommends the use of 10%
neutral buffered formalin to preserve cell morphology as part of the specimen collection
protocol. Basti et al2, described this procedure as a method for managing >100 lobster
specimens during a study on post-capture survivability. The goal of this two part study was to
first compare the total WBC counts from freshly collected elasmobranch (sharks, stingrays)
blood with an aliquot of the same sample that was preserved in formalin. The second part
extended the comparison to include WBC, RBC and thrombocyte counts from a teleost species;
the striped bass, Morone saxatilis, is a popular sport fish in the Chesapeake Bay. Part 2 also
included two time points, where the same preserved blood samples were analyzed within a week
of collection and again approximately a month later to test an extended sample stability period.
Part 1:
Whole blood was collected during routine examinations from 35 animals representing 11 species
of elasmobranchs housed at the National Aquarium. An aliquot of anticoagulated blood was
preserved in 10% neutral buffered formalin at 1:5. Total WBC counts by hemacytometer were
performed using Natt-Herrick diluent3, adjusted to ~1000 mOsm/kg, on fresh cells within 1 hour
of collection and on preserved cells stored at least 24 hours. The maximum interval between
collection and processing for the preserved cells was 56 days. Following guidelines for method
comparisons in human medicine4, the total allowable error (TEa) for WBC is target ± 15%; for
this study, target equals the mean for the 35 samples (31,000/µL; range 7,150-100,776/µL).
Linear regression calculations for the paired data yielded the correlation coefficient r = 0.99,
y=0.9778x + 382.78 and t-test showed no significant difference (P>0.05). The replication
experiment for random error, where a single sample was counted 20 times by both methods,
resulted in CVfresh = 5.31% and CVpreserved = 4.11%. Therefore, the TEcalc = SEmean + 2CVpreserved,
or 1.95% + 8.22% = 10.17%. Because TEcalc < TEa, the proposed method was judged to be an
acceptable alternative when fresh blood cell counts are not practical.
Part 2:
Following the protocol described for Part 1, without the osmolality adjustment, blood was
collected from 50 wild-caught striped bass for WBC counts, as well as RBC and thrombocyte
counts by standard manual hematology methods. The TEa for RBC is target ± 6% and for
platelets (thrombocytes) is target ± 25%4. Cell counts were performed on the fresh specimens
the day of collection and the mean values for served as the target value for the total error
calculations in the comparison studies. The formalin preserved specimens were stored at
ambient temperature until processed: T1: 1-7 days and T2: 40-50 days. Replication experiments
for WBC (all species), RBC and thrombocytes (bass) were performed by 20 replicate counts
from a single preserved blood specimen to determine the mean, sd and CV%. The CV% was
compared to the precision data from BD Unopette product inserts5 (hemacytometer counts for
human cells) where WBC = 9.4%; RBC = 3.5% and thrombocytes (platelets) = 5.9%. The CV%
for the bass cells were WBC = 4.23%, RBC = 9.46% and thrombocytes = 7.26%.
WBC results for T1 (T2): Linear regression calculations for the paired data yielded the correlation
coefficient r = 0.94 (r = 0.98), y=0.951x + 675.16 (y=1.018x – 92.08) and t-test showed no
significant difference (P> 0.05) for both time points. TEcalc = 10.41% (9.34%); both less than
TEa of 15% and therefore acceptable. RBC results for T1 (T2): Linear regression calculations for
the paired data yielded the correlation coefficient r = 0.82 (r = 0.74), y=0.670x + 1.13 (y=0.791x
+ 0.51) and t-test showed no significant difference (P> 0.05) for T1, however P>0.05 for T2.
TEcalc = 20.86% (25.59%); both far greater than the TEa of 6%, therefore the RBC tests failed.
Thrombocyte results for T1 (T2): Linear regression calculations for the paired data yielded the
correlation coefficient r = 0.89 (r = 0.88), y=0.953x + 2738.1 (y=0.803x + 8341.5) and t-test
showed no significant difference (P> 0.05) for both time points. TEcalc = 16.25% (16.32%); both
less than TEa of 25% and therefore acceptable.
Conclusions
The tests for WBC (all species) and thrombocytes resulted in calculated total error less than the
goal as established for human cells. In contrast, the large CV% for the RBC nearly three times
that for human cells, lead to a marked increase in the calculated total error, far exceeding the 6%
goal. Interestingly, the CV% reported for the sandbar shark was comparable to that observed for
the bass in this study (9.2% and 9.46%, respectively)1. This suggests that a greater imprecision
may be inherent for cell counts for non-mammalian erythrocytes (oval cells that retain the
nucleus).
This technique will benefit hematology studies of fish species by extending the specimen
stability period for WBC and thrombocyte counts, which are especially for useful for large field
studies. Further consideration of RBC total allowable error for non-mammalian species is
needed to determine if this is a useful test for health assessment.
References
1. Arnold JE. Hematology of the sandbar shark, Carcharhinus plumbeus,: standardization of
complete blood count techniques for elasmobranchs. Vet Clin Pathol. 2005; 34 (2):115123.
2. Basti D, Bricknell I, Hoyt K, Change ES, Halteman W, Bouchard D. Factors affecting
post-capture survivability of lobster Homarus americanus. Dis Aquat Org. 2010; 90:153166
3. Natt MP, Herrick CA. A new blood diluent for counting the erythrocytes and leucocytes
of the chicken. Poultry Sci. 1952; 31(4):735-738.
4. CLIA-88 Proficiency testing criteria for acceptable performance. Federal Register
February 28, 1992; 57 (40): 7002-7186. Also http://www.westgard.com/clia.htm;
accessed August 14, 2013.
5. Becton Dickinson Corporation, Franklin Lakes, NJ; product inserts for Unopette 5850
(WBC, Platelets) and 5853 (RBC).