Abstract
The goal of the authors of this manual was to present standard haematology procedures for the morphological examination of the cellular elements of blood and bone marrow. The general protocols provided should allow the study of other haematopoietic tissues in research protocols, as well as in mice that are different from the standard male and female C57BL/6J and BALB/cJ species, thus contributing to better understanding of the haematology of mouse models of human disease. A reason behind the publication of this manual might have been the observation by the authors that the scientific interpretation of peripheral blood basophilia in allergy models in mice, based on flow cytometric marker identification, appeared to conflict with microscopic interpretation of such cells based on their conventional Romanowsky type staining characteristics.
This manual describes basic technical haematology procedures, including red and white blood cell counts, platelet counts, slide and smear preparation and staining, and microscopic cell differentiation. Protocols include Material, Methods, Troubleshooting, and, if indicated, Discussion sections. Some techniques are illustrated in short video clips on a DVD. This is included with the book. In addition, some of the numerous colour photographs to illustrate microscopic normal white cell morphology of peripheral blood and bone marrow are also reproduced on a poster entitled Mouse Peripheral Blood Cells. The poster also contains brief descriptions of each cell type, including cell counts and haematological staining morphology, and other haematological and physiological parameters such as haematocrit, total blood volume, blood pressure and heart rate. The source materials were male and female C57BL/6J and BALB/cJ mice.
The content of the manual is divided into six technical chapters, followed by a chapter with conclusions, a list of references and a chapter on laboratory safety aspects when working with certain reagents needed for staining and counting of cells. A two-page index concludes this book. Chapter 1 addresses the specifics of collection of peripheral blood from the tail vein or the tip of the tail in live mice. This is illustrated in two short video clips (Video 1: Venous Access for Blood Film Using Lateral Tail Vein; Video 2: Venous Access for Blood Film Using Tip of Tail). In Chapter 2 (4 protocols), the counting of red blood cells, platelets and viable nucleated white blood cells is described using unopettes and a haemacytometer. This includes methods for determining the percent packed red cell volume adapted to very small blood samples. Chapter 3 (3 protocols) gives detailed instructions on how to make high-quality blood smears on previously cleansed slides and their fixing, staining and mounting for microscopic evaluation and storage (Video 3: Blood Film; Video 4: Staining with Coplin Jar or Carriages; Video 5: Coverslip). Finally, the preparation of cytospins is explained and also illustrated in a video (Video 6: Cytocentrifuge Procedures). Chapter 4 then devotes considerable space to the cell differential assessments of peripheral blood films, illustrated by numerous colour plates in the book and also on a poster. The last two chapters are focused entirely on bone marrow. Chapter 5 (3 protocols) elaborates on the preparation of bone marrow for microscopic examination, including protocols for the actual biopsy of bone marrow, as well as preparing brush smears or cytospins (Video 7: Surgical Exposure of Femur; Video 8: Brush Smear of Marrow). Chapter 6 then covers the cell differential assessments of bone marrow, illustrated by numerous colour photographs.
This state-of-the-art haematology manual outlining standard practices as they specifically apply to mice has a certain merit in any laboratory unfamiliar with standard haematology practices and performing analysis of haematology parameters in mice. However, there are some deficiencies in this book that limit its value. These include the lack of protocols for collection of blood in dead animals, such as the heart and major blood vessels. Furthermore, as a consequence of the exclusive focus on normal cell properties, any reference to the understanding of haematological concepts is absent in this book. Since the blood cells react even to repeat blood sampling, it would have been helpful to find an outline of some of the most prominent changes in quantity and quality of blood cells and the significance of parameters illustrating such concepts. These include a protocol for staining reticulocytes in peripheral blood or the use of the Myeloid:Erythroid ratio (M:E ratio) in bone marrow analysis. Likewise, as the authors themselves acknowledge, new technologies such as flow cytometry have not been addressed in this manual. This is unfortunate since the new generation of veterinary haematology instruments provides species-specific measurement modes, based on flow cytometry, including those for mice. These instruments are capable of analysing as little as 40 μL of blood, they save time and also allow good reproducibility of haematology data. Such new technologies had they been included, could have contributed to a better understanding of apparent inconsistencies with traditional microscopic appearance of certain cell types such as the very rare peripheral basophilic granulocyte in mice. Finally, standard veterinary haematology texts such as the most recent edition of Schalm's Veterinary Hematology 1 that provides valuable general haematology concepts and reference ranges as well as numerous helpful references for mice haematology is not included in the reference list.