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RESEARCH COMMUNICATION

Improved Mouse Blood Smears Using the DiffSpin Slide Spinner

Kirsten Wilkinson, MS, MT (ASCP); James Fikes, DVM, PhD; Susan Wojcik, DVM, PhD

Abstract: Push smears of mouse blood prepared for differential white blood cell (WBC) determination often have many lysed WBCs, numerous RBC "ghosts", and poor morphology of intact RBCs. The purpose of this study was to compare the quality of peripheral blood smears prepared by 3 different methods and to optimize a technique for mouse blood differential WBC determination. Peripheral blood smears were prepared from blood obtained from clinically normal adult mice and human adults. Differential WBC counts, numbers of lysed WBCs/100 intact WBCs, and RBC morphology were compared in blood smears made using the standard push method with undiluted blood, the push method with blood diluted 1:5 with bovine serum albumin, and in centrifugally-prepared smears made with the DiffSpin Slide Spinner (StatSpin, Norwood, Mass, USA). The number of damaged WBCs in mouse versus human samples using the push method was compared using an unpaired Student's t test. ANOVA was used to compare differences in WBC differential counts and numbers of damaged WBCs among the 3 methods for each species. In addition, unpaired Student's t tests were used to compare each method against the other methods, within species. The number of damaged WBCs/100 intact WBCs was approximately 3 times higher in mouse than in human push smears (P = .002). There was no significant difference in WBC differential cell counts among the 3 methods in either species. However, compared with both push techniques, a significantly (P < .01) greater number of intact cells was observed with the DiffSpin technique for mouse blood samples (damaged WBC/100 intact cells = 4.4 ± 2.6 for DiffSpin smears, 9.5 ± 3.9 for push smears with added albumin, and 31.3 ± 10.2 for standard push smears). DiffSpin mouse blood smears consistently had better RBC morphology when compared with standard push smears. In conclusion, the DiffSpin Slide Spinner produced optimal smears of mouse blood for WBC differential determination and analysis of RBC morphology. (Vet Clin Pathol. 2001;30:197-200) ©2001 American Society for Veterinary Clinical Pathology Key Words: Blood smear, DiffSpin, hematology, methodology, mouse

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In our laboratory, all mouse differential WBC counts are performed manually, because a previous method comparison demonstrated unacceptable differences between the results of manual differentials and those produced by the 5-part differential of our automated CBC analyzer (Cell Dyn 3500, Abbott Laboratories, Abbott Park, Ill) (K.W., unpublished data, 1999). In most cases, when making a push or a wedge smear of human blood, relatively few cells are damaged, such that a large number of smudge cells on a human smear may be indicative of disease.1 In contrast, the same smear method used in our laboratory with normal mouse blood results in a significant number of damaged WBCs, including smudge cells and naked nuclei, as well as numerous RBC ghosts and distorted RBCs. As a result, the technologist spends extra time finding 100 intact WBCs for a differential count. Because of the damaged WBCs, RBC ghosts, and RBC distortion, the accuracy of the differential and smear evaluation becomes questionable. In this study, we compared the quality of peripheral blood smears prepared by 3 different methods to optimize a technique for mouse blood differential WBC determination. Peripheral blood smears were prepared using the push method with undiluted blood, the push method with blood diluted 1:5 with 22% bovine serum albumin (BSA), and the DiffSpin Slide Spinner (StatSpin, Norwood, Mass, USA).The addition of 22% BSA to human peripheral blood specimens has been reported to minimize WBC disintegration.2 The DiffSpin Slide Spinner is an instrument that centrifugally generates blood smears. High quality blood smears with a uniformly distributed monolayer have been prepared with human blood using this instrument; and the quality of the blood smears is independent of operator skill.3

From the Department of Preclinical Development, Human Genome Sciences Inc, Rockville, Md. Corresponding author: Kirsten Wilkinson, MS, MT (ASCP), Department of Preclinical Development, Human Genome Sciences Inc, Rockville, MD 20850 (e-mail: [email protected]).

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Table 1. Mean (SD) differential WBC counts and the number of damaged WBCs per 100 intact WBCs in mouse and human blood smears prepared by the push method, with and without bovine serum albumin (BSA), and by the DiffSpin Slide Spinner. Parameter Push

Monocytes (%) Lymphocytes (%) Neutrophils (%) Bands (%) Variant Lymphocytes (%) Eosinophils (%) Basophils (%) Damaged WBCs/100 intact WBCs 4.7 (2.4) 79.7 (8.1) 10.5 (6.4) 2.3 (2.0) 1.2 (1.5) 1.6 (1.4) 0.0 (0.0) 31.3 (10.2)*

Mouse (n = 10) Push + BSA

3.9 (1.8) 82.8 (5.6) 9.4 (4.2) 1.7 (1.3) 1.1 (0.9) 1.1 (1.1) 0.0 (0.0) 9.5 (3.9)

DiffSpin

3.0 (1.9) 82.3 (6.9) 10.2 (5.2) 1.6 (1.2) 1.3 (2.1) 1.6 (1.4) 0.0 (0.0) 4.4 (2.6)

Push

8.8 (4.0) 38.6 (11.2) 50.2 (12.2) 0.0 (0.0) 0.0 (0.0) 1.4 (1.1) 1.0 (1.0) 11.8 (7.1)

Human (n = 5) Push + BSA

6.8 (5.0) 37.6 (12.9) 52.2 (13.2) 0.2 (.0.4) 0.0 (0.0) 2.6 (2.4) 0.6 (0.5) 5.0 (3.4)

DiffSpin

7.6 (5.1) 34.0 (11.5) 53.4 (12.0) 0.2 (0.4) 0.4 (0.9) 3.4 (2.3) 1.0 (1.2) 1.6 (1.1)§

* Significantly different from human push smear technique, P=.0022. Significantly different from mouse push smear technique, P<.0001. Significantly different from mouse push smear (P<.0001) and mouse push smear + BSA techniques, P=.0031. § Significantly different from human push smear technique, P=.0133.

Materials and Methods

Mice were housed and utilized according to an Institutional Animal Care and Use Committee-approved protocol. Normal adult female C57BL/6 mice (n = 15; Harlan, Indianapolis, Ind) were anesthetized with CO2 and the posterior vena cava was exposed. Blood was obtained from the posterior vena cava using a 1-mL syringe (Becton Dickinson, Franklin Lakes, NJ) and a 25gauge needle (Becton Dickinson). The needle was removed from the syringe and blood was ejected into EDTA Capiject blood collection tubes (Terumo Medical Corporation, Somerset, NJ) and thoroughly mixed before preparing the blood smears. Blood was obtained with informed consent from clinically normal adult female (n = 3) and male (n = 2) human volunteers. Blood was collected from the median cubital vein using a Vacutainer blood collection system and EDTA Vacutainer tubes (Becton Dickinson). The first 5 mouse blood specimens were used to optimize the DiffSpin technique for mouse blood. Optimization of the DiffSpin smear technique was necessary because the instrument settings, volume of blood applied, and surface tension of the slide affect the cell density of the smear.3 The DiffSpin Slide Spinner has 5 acceleration settings and 5 duration settings, each designated A through E. The DiffSpin slide holder has 2 holes on its surface for the blood to be placed on the slide, 1 toward the edge of the slide and 1 toward the middle. Mouse blood smears were made with volumes of blood ranging from 20 to 50 µL added to 1 or both of the holes on the slide holder, and with different combi-

nations of acceleration and duration on both Gold Seal Rite-On microscope slides (Gold Seal Products, Portsmouth, NH) and Fisherbrand Superfrost Microscope Slides (Fisher Scientific, Pittsburgh, Penn). Smears were examined for cell spread, artifact, and general morphology. Once the optimal procedure for mouse blood DiffSpin smears was determined, 2 DiffSpin smears, 2 push smears, and 2 push smears made with the addition of 5 µL of 22% BSA (Sigma Chemical Co., St. Louis, Mo) to 25 µL blood were prepared from each of the remaining 10 mouse blood specimens and the 5 human blood specimens. DiffSpin smears of human blood were made with 30 µL of blood at intermediate duration (setting "C") and acceleration (setting "C") on Gold Seal slides. Smears were made within 1 hour of blood collection. All smears were air dried and stained within 1 hour on a Hematek 1000 slide stainer (Bayer Corp, Diagnostics Division, Elkhart, Ind) with a modified Wright-Giemsa Stain Pak (Bayer Corp). Smears were examined in an unblinded manner by a single technologist (K.W.) at 400 magnification. Differential WBC counts were done by counting 100 consecutive intact WBCs on each smear. At the same time, the number of damaged WBCs/100 intact WBCs was determined and a subjective analysis of RBC morphology was made. Data were analyzed using an unpaired Student's t test to compare the number of damaged WBCs between mouse and human push smears. Differential WBC counts and numbers of damaged WBCs were compared using ANOVA to determine whether there was a statistical difference (P < .05) among the 3 smear techniques

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Wilkinson, Fikes, Wojcik

A

C

B

D

Figure 1. Mouse and human blood smears prepared with different methods. (A) Human blood, push method. (B) Mouse blood, push method. Note the "naked" WBC nucleus in the lower right and numerous RBC ghosts (C). Mouse blood, push method with albumin. Note the RBC artifacts. (D) Mouse blood, DiffSpin method. Note the intact WBCs and excellent RBC morphology. All mouse smears were made from the same sample. Modified Wright-Giemsa, 400. for each species. Unpaired t-tests were used to determine whether there were statistical differences (P < .05) in the number of damaged WBCs for each method versus the other methods, within species. mouse or human samples (Table 1). The number of damaged WBCs/100 intact WBCs was approximately 3 times higher in mouse than in human blood push smears (P = .0022). There was a significant difference in the number of damaged cells observed with the 3 smear methods in both mouse (P < .0001) and human (P = .0130) samples. The number of damaged cells in DiffSpin smears was significantly less than that in push smears for both mouse (P < .0001) and human (P = .0133) samples, and was significantly less than that in push smears with BSA in the mouse (P = .0031). Mouse push smears had many smudged RBCs and RBC "ghosts" while human push smears had excellent RBC morphology (Figure 1). Mouse push smears with albumin had fewer RBC ghosts than standard push smears, but had a moderate amount of RBC artifact. Mouse DiffSpin smears had excellent RBC morphology with almost no RBC ghosts. Not all areas of the DiffSpin smear were suitable for evaluation. However, with mouse blood, the cellular distribution and morphology

Results

Before comparing WBC parameters among the 3 methods of blood smear preparation, optimal settings for mouse blood smear preparation with the DiffSpin were determined. The longest duration setting ("E") and highest acceleration setting ("E") produced the best smears for mouse blood samples based on subjective observation of cell spread and morphology. A blood volume of 25 µL placed in the hole closest to the edge of the slide on the slide holder was sufficient to produce 1 smear. The Gold Seal slides were better for DiffSpin smears than were the Fisherbrand slides. There were no significant differences in WBC differential results among the 3 smear methods for either

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in the best areas of the DiffSpin smear were much better than those in the best areas of the push smear.

Discussion

In this study, the preparation of mouse blood smears using the DiffSpin Slide Spinner was optimized to produce high quality blood smears. Although the method of preparation of mouse blood smears did not significantly affect the percentages of WBCs in the peripheral blood, smears prepared with the DiffSpin Slide Spinner using the optimized procedure produced fewer damaged WBCs and better RBC morphology. Based on this experiment and our previous experience, mouse blood cells appear to be more susceptible to mechanical damage in the smear-making process than are human blood cells. Thus, the standard push technique, which produces shearing and pressure forces on blood cells, may not be the most appropriate method for preparing smears of mouse blood. Because cellular damage in mouse blood smears is reduced using the DiffSpin Slide Spinner, fewer total cells must be scanned to evaluate 100 intact WBCs, and a significant amount of technologist time is saved. RBC morphologic changes also are easier to identify. As with a standard push smear, the technologist still must identi-

fy an area in DiffSpin smears with appropriate cellular distribution for examination. With the DiffSpin, highquality mouse blood films can be produced independent of individual technical ability. This advantage is particularly important in research facilities that send out blood samples to clinical pathology reference laboratories, because a high quality smear made on-site with fresh blood is preferable to a smear made hours later at the reference laboratory. In conclusion, we found the DiffSpin Slide Spinner to be an outstanding tool for preparing mouse blood smears. Because of its use in our laboratory, we have greater confidence in the results of our mouse WBC differential counts. It is easy to use and makes smears of consistently excellent quality. 9

Acknowledgment

The authors thank Steve Strawn for help with statistical analysis.

References

1. Glassy EF, ed. Color Atlas of Hematology. Northfield, Ill: College of American Pathologists; 1998:286-287. 2. Densmore CM. Eliminating disintegrated cells on hematologic smears. Lab Med. 1981;12:640-641. 3. StatSpin. Operators Manual for DiffSpin Slide Spinner Model DSO2. Norwood, Mass: StatSpin; 1995.

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