Received: 14 July 2022 | Revised: 21 February 2023 | Accepted: 4 April 2023 DOI: 10.1111/vcp.13258 ORIGINAL ARTICLE Effect of storage on the nitro blue tetrazolium reduction test in dog blood samples Marta Baxarias | Laia Solano-­Gallego Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Abstract Universitat Autònoma de Barcelona, Introduction: The nitro blue tetrazolium (NBT) reduction test (NBTT) has been used Bellaterra, Spain for measuring the metabolic activity of phagocytes of mammals. Activated neutro- Correspondence phils transform NBT into formazan in the cytoplasm. The NBTT can detect the ac- Laia Solano-­Gallego, Departament de Medicina i Cirurgia Animals, Facultat de tivation of neutrophils in peripheral blood and is used to assess neutrophil function Veterinària, Universitat Autònoma de in dogs. However, the NBTT is not used frequently in the clinical setting, as samples Barcelona, Bellaterra, Spain. Email: laia.solano@uab.cat should be processed after blood collection. Objective: The aim of this study was to evaluate the effect of storage on NBTT in dog blood samples. Materials and Methods: Residual EDTA blood samples from 22 dogs were included of different ages, breeds, and sex. The buffy coat layer was separated from the blood and incubated with 0.1% NBT. The NBTT was performed at 0, 24, 48, and 72 h after the collection of blood. Blood samples were stored at 4°C until the tests were per- formed. Blood smears were evaluated by light microscopy, and the NBT reduction rate was reported, which represents the percentage of activated neutrophils. The NBT reduction rate was calculated after counting 300 neutrophils in each slide. Results: The means of NBTT in dog blood samples at 0, 24, 48, and 72 h were 8.3%, 8.5%, 8.7%, and 7.8%, respectively. No significant differences were observed be- tween time points. Conclusions: This study showed that the NBTT can be performed up to 72 h after the collection of canine blood if correctly refrigerated at 4°C. This finding supports the performance of the NBTT in the clinical setting. KEYWORDS activated neutrophils, canine, formazan, metabolic measurement, NBT 1 | I NTRO D U C TI O N can be transformed into insoluble and blue-­colored formazan when reduced.1 Consequently, the reduction of NBT has been used to The nitro blue tetrazolium reduction test (NBTT) has been widely measure reactive oxygen species (ROS) production in phagocytes.1 used for measuring the metabolic activity of mammals and microbial For example, activated neutrophils and monocytes can re- cells.1–­3 The NBT is a soluble and colorless ditetrazolium salt that duce NBT into formazan inside the phagocytic vacuole by the This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2023 The Authors. Veterinary Clinical Pathology published by Wiley Periodicals LLC on behalf of American Society for Veterinary Clinical Pathology. Vet Clin Pathol. 2023;00:1–5.  wileyonlinelibrary.com/journal/vcp | 1 | 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13258 by Readcube (Labtiva Inc.), Wiley Online Library on [03/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2 BAXARIAS and SOLANO-­G ALLEGO NADPH-­oxidase enzyme,1–­3 and the amount of reduced NBT is di- 2.2 | Residual EDTA blood storage and nitro blue rectly proportional to the amount of ROS produced in the oxidative tetrazolium reduction test burst.4 Then, the NBT reduction rate can be obtained by calculating the percentage of neutrophils and monocytes containing formazan After blood collection, residual EDTA blood samples were placed in their cytoplasm by ordinary light microscopy. 2 Thus, NBTT can into separate Eppendorf tubes (around 100 μL in each Eppendorf) detect the activation of neutrophils and monocytes in peripheral within 1 h from sampling. One of the tubes was kept at room tem- blood. perature and immediately used for NBTT, while the other three were Furthermore, this test has been previously used to assess canine stored at 4°C until further use at 24, 48, and 72 h, respectively. neutrophil function in leishmaniosis,3 monocytic ehrlichiosis,5 dia- The NBTT was performed as described elsewhere3 with some betes mellitus,6 and transitory immunosuppression following immu- modifications. Blood was left at room temperature (between 20°C nization with polyvalent vaccines.7 However, the NBTT is not used and 25°C) for 15 min before NBTT was performed. Afterward, blood frequently in veterinary clinical settings, probably due to the assay's was mildly mixed, and three 40 mm/20 μL hematocrit capillary mi- protocol limitations which dictate that the samples should be pro- crotubes (Servopax, Wiesel, Germany) were filled. Then, microtubes 8 cessed within 2–­6 h after sample collection. were centrifuged at 2910 g for 5 min (Fugevet+ GDC005, Nahita Hence, the aim of this study was to evaluate the effect of storage International LTD, London, UK) to obtain the buffy coat layer. After at 4°C of the samples up to 72 h on the results of NBTT in dog blood centrifugation, the buffy coat layer from the three microtubes was samples. harvested after carefully breaking the microtubes in half (as near as possible to the buffy coat without producing any damage) and then pushing the buffy coat with a syringe and placing it in an Eppendorf. 2 | M ATE R I A L S A N D M E TH O DS When all buffy coats were placed in an Eppendorf tube, a similar volume of 0.1% NBT (N6876, Sigma-­Aldrich Co., St. Louis, USA) 2.1 | Dogs solution was added (proportion 1:1). The Eppendorf was mildly mixed, and incubated for 15 min in a heater at 37°C, and another Residual EDTA blood samples from 22 dogs were included in this 15 min at room temperature (between 20°C and 25°C). After incu- prospective study. Between one to six milliliters of blood was col- bation, three blood smears were obtained by placing 3 μL of NBT-­ lected from the dogs by jugular or metatarsal venipuncture for rou- stained blood on each slide. Each slide was subsequently stained tine laboratory tests. The dogs were from Catalonia (Spain) and were with Diff-­Q uik (Sigma-­Aldrich Co., ST. Louis, USA) and evaluated sampled in 2019 for an annual health check-­up. The dogs belonged by ordinary light microscopy, first at 10× and 20× magnifications to private owners, and written informed consent was obtained from to scan the slide and at 40× (Figures 1 and 2) and 60× magnifi- the respective owners (including staff and student volunteers). This cation for cell counting, and 100× (Figure 3) magnification during includes consent for the use of any remaining materials from those cell counting to confirm the deposition of formazan in those cells initially obtained for diagnostic purposes. Therefore, ethical study with low deposition that could be confused with artifacts. The test approval was not needed due to the use of residual blood samples. A physical examination was performed for all dogs included in the study. Four dogs were considered sick due to dermatological clini- cal signs compatible with clinical leishmaniosis,9 and they were also seropositive in a quantitative in-­house ELISA for the detection of L. infantum-­specific antibodies.10 Briefly, serum samples were diluted to 1:800 in phosphate-­ ­ buffered saline (PBS)-­ Tween with 1% dry milk and incubated for 1 h at 37°C. After performing three washes with PBS–­Tween and one wash with PBS (pH = 7.3), samples were incubated for 1 h at 37°C with peroxidase-­conjugated Protein A (Peroxidase Conjugate Protein A; Merck KGaA) at a concentration of 0.16 ng/μL. After incubation, washes were repeated, and the o-­phenylenediamine and substrate buffer (SIGMAFAST OPD; Merck KGaA) was added to the plate. The reaction was stopped with 5 M H2SO 4. Results were read at 492 nm in a spectrophotometer machine (MB-­580 HEALES; Shenzhen Huisong Technology Development Co., Ltd, Shenzhen, China) and were defined as ELISA units (EU) in rela- F I G U R E 1 Several neutrophils without reduced formazan in the cytoplasm (red arrows) and another neutrophil/monocyte showing tion to a positive canine sera sample used as a calibrator set at 100 reduced formazan in the cytoplasm (black arrow) surrounded by EU as described elsewhere.10 Dogs were seropositive when having a red blood cells (×40 objective) stained with Diff-­Quik. Formazan result equal to or higher than 35 EU. The other 18 dogs were classi- sometimes hinders differentiation between neutrophils and fied as apparently healthy. monocytes. | 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13258 by Readcube (Labtiva Inc.), Wiley Online Library on [03/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License BAXARIAS and SOLANO-­G ALLEGO 3 F I G U R E 2 Two neutrophils/monocytes showing reduced formazan in the cytoplasm (black arrows) surrounded by red blood cells (×40 objective) stained with Diff-­Quik. Formazan sometimes F I G U R E 4 Change of NBT reduction rate (%) of peripheral blood hinders differentiation between neutrophils and monocytes. neutrophils in each studied time point. The procedure described above was performed at 0 h (fresh blood) and 24, 48, and 72 h (blood stored at 4°C) in 17 blood sam- ples. In the other five cases, the procedure was not performed at 72 h due to a lack of samples. 2.3 | Statistical analysis The statistical analysis was performed using the package Stats for the software R i386 3.6.1 for Windows, using an ANOVA for repeated measures with Dunnett's multiple comparison tests to detect differ- ences between time points (0 h vs 24 h, 48 h, and 72 h) and t-­test was used to compare between groups (crossbreed or purebred, female or male, and healthy or sick). The Shapiro–­Wilk test was performed to detect the normal distribution of quantitative variables. NBT F I G U R E 3 One neutrophil without reduced formazan in the ­reduction rate, sex, and age data were normally distributed (P > 0.05). cytoplasm (red arrow) and another neutrophil/monocyte showing A P-­value of <0.05 was considered statistically significant. Graphs reduced formazan in the cytoplasm (black arrow) surrounded by were plotted using GraphPad Prism version 5.00 for Windows red blood cells (×100 objective) stained with Diff-­Quik. Formazan sometimes hinders differentiation between neutrophils and (GraphPad Software, San Diego, CA, USA). monocytes. results were reported as NBT reduction rate, which represents the 3 | R E S U LT S percentage of activated neutrophils/monocytes. The NBT reduc- tion rate was calculated after counting 300 phagocytes in each slide Most of the dogs were classified as crossbred (72.7%, 16/22), while (which included cells with blue formazan deposits+non-­activated the purebred dogs included two Labrador retrievers, two Golden re- neutrophils). Those cells aggregated or broken were rejected during trievers, one Spanish greyhound, and one Ibizan hound. The mean cell counting. Thus, the percentage was the number of activated age was 6 years and ranged from 1 to 13 years. Both sexes were in- neutrophils/monocytes, as defined by those containing dark blue cluded, with 12 females (63.2%) and 7 males (36.8%). Information on formazan deposits, divided by the total number of non-­activated age and sex was missing for three dogs. neutrophils and multiplied by 100. It is important to highlight that it The mean NBT reduction rate of neutrophils and monocytes at is difficult to differentiate neutrophils from monocytes when there 0, 24, 48, and 72 h was 8.3%, 8.5%, 8.7%, and 7.8%, respectively are blue formazan deposits. (Figure 4). No significant differences were observed between time | 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13258 by Readcube (Labtiva Inc.), Wiley Online Library on [03/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 BAXARIAS and SOLANO-­G ALLEGO TA B L E 1 NBT reduction rate results. Mean NBT (%) reduction rate (±SD) Number of dogs 0h 24 h 48 h 72 h* Total (N = 22) 8.3 (±5.3) 8.5 (±5.4) 8.7 (±4.8) 7.8 (±4.0) Breed Crossbreed (n = 16) 7.7 (±4.7) 7.7 (±4.9) 8.2 (±4.4) 7.1 (±3.7) Purebred (n = 6) 10 (±6.7) 10.6 (±6.6) 10.1 (±6.1) 10.1 (±4.7) Sex** Female (n = 12) 8.2 (±5.6) 8.6 (±6.3) 8.8 (±5.5) 7.6 (±4.5) Male (n = 7) 8.6 (±4.2) 9.5 (±4.0) 9.6 (±3.6) 8.2 (±3.4) Clinical status Apparently healthy (n = 18) 8.5 (±5.6) 8.7 (±5.9) 8.9 (±5.2) 7.9 (±4.4) Sick*** (n = 4) 7.3 (±3.3) 7.4 (±2.7) 7.6 (±2.0) 7.4 (±2.6) *The procedure was not performed at 72 h due to a lack of samples in five dogs.; **Information on sex was missing in three dogs.; ***Four dogs were considered sick due to dermatologic clinical signs compatible with clinical leishmaniosis, and they were also seropositive in a quantitative in-­house ELISA for the detection of L. infantum-­specific antibodies. Abbreviations: NBT, nitro blue tetrazolium; SD, standard deviation. points (repeated measures ANOVA with Dunnett's multiple compar- Interestingly, other factors, such as the choice of anticoagu- ison tests: F = 1.58, P = 0.2055). lant and the type of blood sample (capillary or venous), have been The mean NBT reduction rate depending on breed, sex, and clin- previously investigated when performing the NBTT and have been ical status at 0, 24, 48, and 72 h is shown in Table 1. No differences proven to affect the results.11,23–­25 For example, using heparin as in NBT reduction rates were found when comparing crossbred and an anticoagulant could produce stimulation of oxidative metabo- purebred, female and male, and healthy and sick dogs (P > 0.05). lism and, thus, increase the percentage of activated neutrophils and monocytes in the sample.11,23 Regarding the type of blood sample when comparing capillary and venous blood, Randall et al24 reported 4 | DISCUSSION no differences in the NBT reduction rate between capillary and ve- nous blood in healthy patients. However, in another study, 25 the 11 A previous study performed on human samples described that proportion of activated neutrophils and monocytes was significantly storing blood samples for up to 8 h at 4°C or for up to 4 h at 23°C lower in capillary blood than in venous blood in sick patients with did not influence the test results. Additionally, when blood samples an increased proportion of NBT rate. Unfortunately, in the present were stored at 23°C for 8 h or more, the test results increased, but study, only peripheral blood was collected from dogs and investi- the reasons were not further investigated.11 In the present study, gated with NBTT; thus, it was not possible to investigate the dif- storage of dog blood samples at 4°C was longer, varying from 24 ferences between peripheral and capillary blood in canine samples. to 72 h, and the results were similarly not influenced by the time-­ Furthermore, dog characteristics such as breed, sex, and clinical lapse. We believe that this is the first time that such a result has status do not seem to affect the NBT reduction rate during stor- been reported, as the NBTT protocol demands sample processing age, although more information should be included to confirm these within 2–­6 h after blood collection.8 This finding might enable the results. NBTT to win a place among the standard laboratory tests available Although the present study reports interesting results regarding to the clinician to assess canine neutrophil and monocyte function in NBTT in dogs, some limitations must be considered. Further studies sick dogs.3,5–­7 For example, dogs with canine leishmaniosis present- should include a higher number of dog samples, specifically samples ing a mild disease have a higher NBT reduction rate than healthy from dogs with diseases that could affect the number of activated 3 dogs, and thus, the NBTT could be used to detect improvement in neutrophils and monocytes, such as dogs with canine leishmanio- these cases. In fact, any abnormalities or diseases that affect phago- sis,3 monocytic ehrlichiosis,5 or diabetes mellitus.6 Moreover, fur- cytic activity in both inflammatory and bacterial processes could be ther studies should evaluate if the NBTT can be performed in blood studied using the NBT reduction rate. In human medicine, the NBT samples stored longer than 72 h. In addition, the use of imaging soft- reduction rate has been used in numerous cases such as chronic granu- ware could be helpful assessing the degree of the color change of lomatous diseases,12,13 tuberculosis,14 viral meningoencephalitis,15 activated neutrophils and monocytes in canine blood samples, as and liver abscesses caused by amebiasis.16 Recently, the NBTT has it has been used previously to assess the intensity of ROS produc- also been used in several other cases, such as to investigate the im- tion in mouse spermatozoa by NBTT. 26 Furthermore, non-­activated mune profile of human patients with fungus infection,17 gingival monocytes should have also been included during the cell counting fibromatosis,18 chronic obstructive pulmonary disease,19 and ROS of each slide, even if their exclusion did not affect the results re- production during HL-­60 cell differentiation, 20 in biofilms, 21 and lated to the effect of storage in canine blood samples. Not count- during heat-­stressed whole blood cultures. 22 ing non-­activated monocytes could affect the reported percentage, | 1939165x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/vcp.13258 by Readcube (Labtiva Inc.), Wiley Online Library on [03/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License BAXARIAS and SOLANO-­G ALLEGO 5 10. Solano-­Gallego L, Di Filippo L, Ordeix L, et al. Early reduction of even though the proportion of monocytes in canine blood samples Leishmania infantum-­ specific antibodies and blood parasitemia is usually low, while the proportion of neutrophils is much higher. 27 during treatment in dogs with moderate or severe disease. Parasit Vectors. 2016;9:235. 11. Björksténm B. The influence of technical factors on the NBT test. Scand J Haematol. 1974;12:46-­50. 5 | CO N C LU S I O N 12. Baehner R, Nathan D. Quantitative nitroblue tetrazolium test in chronic granulomatous disease. N Engl J Med. 1968;278:971-­976. This study showed that the NBTT could be performed up to 72 h 13. Chari PS, Chandra P, Prasad S, Bhattad S. Chronic granulomatous after the collection of canine blood if correctly refrigerated at 4°C. disease presenting with small bone osteomyelitis in a young child: a case report. Indian J Pathol Microbiol. 2018;61:557-­560. This finding supports the performance of NBTT in the clinical setting. 14. Dubaniewicz A, Hoppe A. The spontaneous and stimulated ni- troblue tetrazolium (NBT) tests in mononuclear cells of patients AC K N OW L E D G M E N T S with tuberculosis. Rocz Akad Med w Byalymstoku. 2004;49:252-­255. The authors would like to thank Laura Ordeix (ORCID: 15. Kölmel HW, Egri T. The significance of the nitroblue-­tetrazolium test in cerebrospinal fluid granulocytes in bacterial and abacterial 0000-­0 002-­3631-­8358) and Alejandra Álvarez-­Fernández (ORCID: meningitis. Infection. 1980;8:142-­146. 0000-­0 002-­0185-­9138) (Departament de Medicina i Cirurgia Animals, 16. Gandhi BM, Acharya SK, Irshad M, Gupta H, Chawla TC, Twandon Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, BN. Entamoeba histolytica: elevated nitroblue tetrazolium reduction Spain) for the collection of the samples and the subsequent donation activity in polymorphs during amoebic liver abscess. Trans R Soc Trop Med Hyg. 1987;81:283-­285. of residual samples for this study. 17. Zeng Q, Jin Y, Yin G, et al. Peripheral immune profile of children with Talaromyces marneffei infections: a retrospective analysis of 21 C O N FL I C T O F I N T E R E S T S TAT E M E N T cases. BCM Infect Dis. 2021;21:287. The authors have no conflict of interest. 18. Chaturvedi R. Idiopathic gingival fibromatosis associated with gen- eralized aggressive periodontitis: a case report. J Can Dent Assoc. 2009;75:291-­295. ORCID 19. Shanmugam L, Ravinder SS, Johnson P, Padmavathi R, Rajagopalan Marta Baxarias https://orcid.org/0000-0002-1830-4366 B, Kindo AJ. Assessment of phagocytic activity of neutro- Laia Solano-­Gallego https://orcid.org/0000-0001-8479-4896 phils in chronic obstructive pulmonary disease. Lung India. 2015;32:437-­4 40. 20. Huang Q, Wang L, Ran Q, et al. Notopterol-­induced apoptosis and REFERENCES differentiation in human acute myeloid leukemia HL-­60 cells. Drug 1. Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell des Devel Ther. 2019;13:1927-­1940. biology: new insights into their cellular reduction. Biotechnol Annu 21. Marioni J, da Silva MA, Cabrera JL, Núñez Montoya SC, Paraje MG. Rev. 2005;11:127-­152. The anthraquinone rubiadin and its 1-­methyl ether isolated from 2. Gómez-­Ochoa P, Sabate D, Homedes J, Ferrer L. Use of the ni- Heterophyllaea pustulata reduces Candida tropicalis biofilms forma- troblue tetrazolium reduction test for the evaluation of domper- tion. Phytomedicine. 2016;23:1321-­1328. idone effects on the neutrophilic function of healthy dogs. Vet 22. Verma N, Alyethodi RR, Kathuria A, et al. Effect of heat stress on Immunol Immunopathol. 2012;146:97-­99. superoxide anion production in native and crossbred cattle under 3. Gómez-­Ochoa P, Lara A, Couto G, et al. The nitroblue tetrazolium in vitro whole blood culture model. J Therm Biol. 2020;87:102457. reduction test in canine leishmaniosis. Vet Parasitol. 2010;172:​ 23. Hohn DC, Lehrer RI. Mechanism of the heparin effect on the ni- 135-­138. troblue tetrazolium slide test. Infect Immun. 1974;10:772-­775. 4. Muniz-­Junqueira MI, de Paula-­Coelho VN. Meglumine antimon- 24. Randall J, Perriello V, Hendley JO. The NBT test in streptococcal ate directly increases phagocytosis, superoxide anion and TNF-­α pharyngitis. Pediatrics. 1973;51:685-­689. production, but only via TNF-­α it indirectly increases nitric oxide 25. Björkstén B. The NBT test using venous and capillary blood. Scand production by phagocytes of healthy individuals, in vitro. Int J Haematol. 1973;11:270-­274. Immunopharmacol. 2008;8:1633-­1638. 26. Golas A, Malek P, Piasecka M, Styrna J. Sperm mitochondria dia- 5. Hasegawa MY, Kohayagawa A, Brandão LP, Morgulis MSFA, phorase activity—­ a gene mapping study of recombinant inbred Hagiwara MK. Evaluation of neutrophil oxidative metabolism in ca- strains of mice. Int J Dev Biol. 2010;54:667-­673. nine monocytic ehrlichiosis. Vet Clin Pathol. 2005;34:213-­217. 27. Bourgès-­Abella N, Geffré A, Concordet D, Braun JP, Trumel C. 6. Lechowski R, Lenarcik M, Degòrski A, Winnicka A. Serum lysozyme Canine reference intervals for the Sysmex XT-­2000iV hematology activity and nitroblue tetrazolium reduction test in dogs with diabe- analyzer. Vet Clin Pathol. 2011;40:303-­315. tes mellitus. J Vet Med Ser A. 1991;38:530-­533. 7. Strasser A, May B, Teltscher A, Wistrela E, Niedermüller H. Immune modulation following immunization with polyvalent vaccines in dogs. Vet Immunol Immunopathol. 2003;94:113-­121. How to cite this article: Baxarias M, Solano-­Gallego L. Effect 8. Gordon AM, Rowan RM, Brown T, Carson HG. Routine application of the nitroblue tetrazolium test in the clinical laboratory. J Clin of storage on the nitro blue tetrazolium reduction test in dog Pathol. 1973;26:52-­56. blood samples. Vet Clin Pathol. 2023;00:1-5. doi:10.1111/ 9. Solano-­Gallego L, Miró G, Koutinas A, et al. LeishVet guidelines for vcp.13258 the practical management of canine leishmaniosis. Parasit Vectors. 2011;4:86.