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Forgot your password? This atlas is designed for a diverse audience that includes clinical laboratory science students, medical students, residents, and practitioners. It is also a valuable resource for clinical laboratory practitioners who are being retrained or cross-trained in hematology. It is not intended to be a detailed, comprehensive manual for diagnosis. In this concise format, every photomicrograph and word has been evaluated for value to the microscopist. All superfluous information has been excluded in an attempt to maintain focus on significant microscopic findings while correlating this information with clinical diag- nosis.
What started as a primer for Clinical Laboratory Science students with no previous hematology education has evolved into an internationally recognized reference for multiple levels of expertise, from entry level to practicing professionals. Chapter 1 reviews smear preparation, staining, and the appropriate area in which to evaluate cell distribution and morphology. A table that summa- rizes the morphology of leukocytes found in a normal differential, along with multiple exam- ples of each cell type, facilitates early instruction in blood smear review.
Chapter 2 schematically presents hematopoietic features of cell maturation. General cell maturation, along with an electron micrograph with labeled organelles, will help readers cor- relate the substructures with the appearance of cells under light microscopy. Visualizing nor- mal cellular maturation is essential to the understanding of disease processes. This correlation of schematic, electron micrograph, and Wright-stained morphology is carried throughout the maturation chapters.
Figure has been formatted to reflect recent hematopoietic theory. In addition, the chart aids readers in recognizing the anatomical sites at which each stage of mat- uration normally occurs.
Chapters 3 to 9 present the maturation of each cell line individually, repeating the respec- tive segment of the overall hematopoietic scheme from Chapter 2, to assist the student in see- ing the relationship of each cell line to the whole. In these chapters, each maturation stage is presented as a color print, a schematic, and an electron micrograph. A description of each cell, including overall size, nuclear-to-cytoplasmic ratio, morphologic features, and reference ranges in peripheral blood and bone marrow, serves as a convenient summary.
The final figure in each of these chapters summarizes lineage maturation by repeating the hematopoietic seg- ment with the corresponding photomicrographs. Multiple nomenclatures for erythrocyte maturation are used to accommodate use in multiple settings and demographic groups. Chapters 10 to 12 present discrete cellular abnormalities of erythrocytes, that is, variations in size, color, shape, and distribution, as well as inclusions found in erythrocytes.
Each var- iation is presented along with a description of the abnormality, or composition of the inclu- sion, and associated disorders. Because diseases are often combinations of the cellular alterations, Chapter 13 integrates morphologic findings into the diagnostic features of disorders primarily affecting erythrocytes.
Diseases of excessive or altered production of cells may be caused by maturation arrest, asynchronous development, or proliferation of one cell line, as presented in Chapters 15 to Cytochemical stains are presented with disorders in which they are useful. The therapeutic use of myeloid growth factors causes morphologic changes that mimic severe infections or malignancies. Readers are encouraged to refer to the normal hematopoiesis illustration, Figure , for comparison of normal and abnormal cells and the progression of diseases.
Microorganisms, including parasites, may be seen on peripheral blood smears. A brief pho- tographic overview is given in Chapter Chapter 22 includes photomicrographs that are not categorized into any one particular area, such as fat cells, mitotic figures, metastatic tumor cells, and artifacts.
Chapter 23 describes findings expected in the peripheral blood of neonates, including anticipated variations in morphology and cellular distribution. Comparison of the hemato- gone, normal for newborns, with the blast cell of acute leukemia is included. Chapter 24 is intended to be an overview of the most frequent microscopic findings in body fluids.
It is not proposed as a comprehensive review of the cytology of human body fluids, but rather a quick reference for the beginning microscopist as well as the seasoned professional. As with the third edition and fourth editions, the fifth edition features spiral binding, mak- ing the atlas more convenient when used at the microscope bench. All of these chapters combine into what we believe is a comprehensive and valuable resource for any clinical laboratory.
The quality of the schematic illustrations, electron micro- graphs, and color photographs stand for themselves. We hope that this atlas will enrich the learning process for the student and serve as an important reference tool for the practitioner.
Instructors have access to an electronic image collection featuring all of the images from the atlas. Students and instructors have access to summary tables, student review exercises, and additional photos for identification. Bernadette F.
Rodak Jacqueline H. Carla also provided authoritative comments on images and helped us determine which images were classic examples. A particular thank you goes out to our families for their understanding during the many hours that we spent away from them while pursuing this goal. A special thank you goes to the professionals at Elsevier who navigated us through the production of this atlas.
A variety of methods are available for preparing and staining blood films, the most common of which are discussed in this atlas. The wedge film is a con- venient and commonly used technique for making peripheral blood films. High-quality, beveled-edge microscope slides are recommended. One slide serves as the blood film slide, and the other as the spreader slide.
These can then be reversed to prepare a second film. A drop of ethy- lenediaminetetraacetic acid EDTA anticoagulated blood about 3 mm in diameter is placed at one end of the slide. Alternatively, a similar size drop of blood directly from a finger or heel puncture is acceptable.
The size of the drop of blood is important. Too large a drop creates a long or thick film, and too small a drop often makes a short or thin film. In pre- paring the film, the technician holds the pusher slide securely in front of the drop of blood at a to degree angle to the film slide Figure , A.
The pusher slide is pulled back into the drop of blood and held in that position until the blood spreads across the width of the slide Figure , B. It is then quickly and smoothly pushed forward to the end of the film slide, creating a wedge film Figure , C.
It is important that the whole drop of blood is picked up and spread. Moving the pusher slide forward too slowly accentuates poor leu- kocyte distribution by pushing larger cells, such as monocytes and granulocytes, to the very ends and sides of the film.
Maintaining a consistent angle between the slides and an even, gentle pressure is essential. It is frequently necessary to adjust the angle between the slides to produce a satisfactory film. For higher than normal hematocrit, the angle between the slides must be lowered so that the film is not too short and thick. For extremely low hematocrit, the angle must be raised. A well-made peripheral blood film Figure has the following characteristics: 1.
About two-thirds to three-fourths of the length of the slide is covered by the film. It is slightly rounded at the feather edge thin portion , not bullet shaped. Lateral edges of the film should be visible. The use of slides with chamfered beveled cor- ners may facilitate this appearance.
It is smooth without irregularities, holes, or streaks. The whole drop is picked up and spread. Figure shows examples of unacceptable films. A, Correct angle to hold spreader slide. B, Blood spread across width of slide. C, Completed wedge film. From Keohane E. Louis: Saunders Elsevier.
Slide appearances associated with the most common errors are shown, but note that a combination of causes may be responsible for unacceptable films. A, Chipped or rough edge on spreader slide. B, Hesitation in forward motion of spreader slide. C, Spreader slide pushed too quickly.
D, Drop of blood too small. E, Drop of blood not allowed to spread across the width of the slide. F, Dirt or grease on the slide; may also be caused by elevated lipids in the blood specimen.
G, Uneven pressure on the spreader slide. H, Time delay; drop of blood began to dry. Wright or Wright-Giemsa stains are the most commonly used for peripheral blood and bone marrow films.
These stains contain both eosin and methylene blue and are therefore termed polychrome stains. The colors vary slightly from laboratory to laboratory, depending on the method of staining.
Slides must be allowed to dry thoroughly before staining. The cells are fixed to the glass slide by the methanol in the stain. Staining reactions are pH dependent, and the actual staining of the cellular components occurs when a buffer pH 6. Free methylene blue is basic and stains acidic cellular components, such as RNA, blue. Free eosin is acidic and stains basic components, such as hemoglobin or eosinophilic granules, red. Neutrophils have cytoplasmic granules that have a neutral pH and accept some character- istics from both stains.
Details for specific methods of staining peripheral blood and bone marrow films, including automated methods, may be found in a standard textbook of hematology. An optimally stained film Figure has the following characteristics: 1. The red blood cells RBCs should be pink to salmon. Nuclei are dark blue to purple. Cytoplasmic granules of neutrophils are lavender to lilac.
Cytoplasmic granules of basophils are dark blue to black. Cytoplasmic granules of eosinophils are red to orange. The area between the cells should be colorless, clean, and free of precipitated stain. A well-stained slide is necessary for accurate interpretation of cellular morphology. The best staining results are obtained from freshly made slides that have been prepared within 2 to 3 hours of blood collection.
Box lists common reasons for poorly stained slides and may be used as a guide when troubleshooting. FIGURE 1—4 Optimally stained peripheral blood film demonstrating the appropriate area in which to perform the white blood cell differential and morphology assessment and the platelet estimate. If the latter exists, another film should be prepared. Scan eight to ten fields in this area of the film, and determine the average number of white blood cells WBCs per field.
This estimate is a useful quality-control tool for validating WBC counts from hematology analyzers. Any discrepancy between the instrument WBC count and the slide estimate must be resolved. When the correct area of the film from a patient with a normal RBC count is viewed, about to RBCs per oil immersion field are seen see Figure Characteristically, the differential count includes counting and classifying consecutive WBCs and reporting these classes as percentages.
The results are reported as percentages of each type of WBC seen during the count. See Chapter 9 for more detailed information on lymphocyte size. Each laboratory should have established protocols for standardized reporting of abnormalities.
Evaluation of the RBC morphology is an important aspect of the film evaluation and is used in conjunction with the RBC indices to describe cells as normal or abnormal in size, shape, and color. Each laboratory should establish a standard reporting protocol. The microscopic evaluation of RBC morphology must be congruent with the information given by the automated hematology analyzer.
If not, discrepancies must be resolved before reporting patient results. The final step in the performance of the differential count is the estimation of the platelet number. In an area of the film where RBCs barely touch, the number of platelets in five to ten oil immersion fields is counted. The average number of platelets is multiplied by 20, to provide an estimate of the total number of platelets per cubic millimeter.
This estimate is reported as adequate if the estimate is con- sistent with a normal platelet count, decreased if below the lower limit of normal for that laboratory, and increased if above the upper limit of normal. When a patient is extremely anemic or has ery- throcytosis, a more involved formula for platelet estimates may be used. The estimate can be compared with an automated platelet count as an additional quality- control measure.
If the estimate and the instrument platelet count do not agree, discrepancies must be resolved. Some causes for discrepancies include the presence of giant platelets, many schistocytes red blood cell fragments , and platelet satellitism.
SUMMARY A considerable amount of valuable information can be obtained from properly prepared, stained, and evaluated peripheral blood films. WBC differential and morphology and the RBC morphology and platelet estimate are included in the film evaluation. The process begins with the plur- ipotential hematopoietic stem cell multipotent progenitor , which is capable of proliferation, replication, and differentiation. In response to cytokines growth factors , the pluripotential stem cell will differentiate into a common myeloid or common lymphoid progenitor.
Both the myeloid and lymphoid progenitors maintain their pluripotential capac- ity. The lymphoid progenitor proliferates and differentiates into T, B, and natural killer cells. The myeloid progenitor proliferates and differentiates into granulocyte, monocyte, erythrocyte, and megakaryocyte lineages. To this point in maturation, none of these stem cells can be morphologically identified, although it is postulated that they appear similar to a small resting lymphocyte. The blue shaded area in Figure highlights the stem cell populations.
Each lineage and maturation stage will be presented in detail in the following chapters. Hematopoiesis is a dynamic continuum.
Cells gradually mature from one stage to the next and may be between stages when viewed through the microscope. In general, the cell is then identified as the more mature stage. General morphological changes in blood cell maturation are demonstrated in Figure Figures , A and B, illustrate cell ultrastructure. A review of organelles will facilitate cor- relation of morphological maturation with cell function.
Table delineates the location, appearance, and function of individual organelles. A, Cell diameter decreases and cytoplasm becomes less basophilic. B, Nuclear diameter decreases N:C ratio decreases.
Nuclear color changes from purplish red to dark blue. C, Nuclear chromatin becomes coarser, clumped, and condensed. Granules appear in cytoplasm see Chapter 5. D, Composite of changes during maturation process. E, Representative cells from the erythroid series, demonstrating maturation changes. Modified from Diggs, L. The morphology of human blood cells. Abbott Park: Abbott Laboratories.
B, Electron micrograph with labeled organelles. A, From Keohane E. Rodak's hematology: clinical principles and applications. The blue color of the cytoplasm is becoming gray-blue as hemoglobin is produced. The gray-blue color of the cytoplasm is becoming salmon as more hemoglobin is produced. Note that the hemoglobinization. SIZE: 8 to 8.
Megakaryocytes are among the largest cells in the body and mature by a unique process called endomitosis. In endomitosis, the nucleus is duplicated but there is no cell division, resulting in a polyploid cell. Megakaryocyte nuclei may have from 2 to 32 lobes and in unusual cases may have up to 64 lobes. Megakaryocytes develop copious cytoplasm, which differentiates into platelets. Platelets have several types of granules that can be visualized by electron microscopy.
The granules are highly specialized. Identification by morphology alone is not advisable. B, Promegakaryocyte.
C, Megakaryocyte. D, Platelet. E hematopoietic stem cell Each of these divides and matures into cells M known as blasts, one for each cell line. How- ever, it is not possible to differentiate the various blasts at the light microscope level.
P R This chapter addresses neutrophil maturation. Common myeloid O See Chapter 7 for discussion of eosinophils G progenitor and Chapter 8 for discussion of basophils. E As the cells mature from the myeloblast N I to the promyelocyte, there is a slight increase T Granulocyte-monocyte in size, in contrast with size variation in O progenitor other cell lineages. At the promyelocyte R stage, the chromatin in the nucleus becomes slightly coarser than the myeloblast, and pri- mary burgundy-colored azurophilic granules Myeloblast appear in the cytoplasm.
As the cell divides and matures to a myelocyte, chromatin becomes coarser and condensed, and secondary spe- cific granules appear in the cytoplasm begin- P ning at the Golgi apparatus and spreading R Promyelocyte throughout the cytoplasm. Primary granules E are still present but less visible on a Wright- C U stained smear because of a chemical change R Myelocyte in the membranes. It is often possible at the S myelocyte stage to see the area of the Golgi O apparatus, which appears as a clearing close to R the nucleus.
The specific secondary granules Metamyelocyte differentiate the cell into neutrophil, eosino- phil, and basophil. The nucleus then begins to indent and chromatin becomes coarser, sig- naling the metamyelocyte stage.
Finally, the cell becomes a seg- L mented neutrophil when the nucleus becomes segmented or lobated into two to five lobes. The lobes are connected by threadlike fila- ments with no chromatin visible in the filament.
The dynamic nature of maturation is easily seen in the neutrophilic series; that is, cell maturation does not proceed in a stepwise fashion but occurs gradually from one stage to another. Thus morphologically, a cell may appear as a late promyelocyte or an early myelocyte. When there is a question of maturation stage, it is generally preferable to classify the cell at the more mature stage. They give the cytoplasm a grainy or sandy appearance, and the overall color is lavender to pink.
See Figure 7—2 for eosinophilic myelocyte. Dotted line A indicates hypothetical round nucleus. See Figure 7—4 for eosinophilic metamyelocyte. Blood cell identification. In Hematology and clinical microscopy glossary. Specimens for electron microscopy are prepared by embedding tissue in a suitable medium, Refer to Table 1—1 for more examples. Ultra-thin cross sections are then prepared. Because this image shows a cross section, the lobes of the nucleus appear to be separate, but are not.
Rarely, seen in the peripheral blood during severe sepsis. NOTE: Eosinophils are fragile and may easily fracture when preparing blood film. Maturation parallels that of the neutrophil; however, immature stages are very rare and generally seen only in basophil proliferative disorders. Note that granules are water- soluble and may be dissolved during the staining process, leaving clear areas in the cytoplasm.
Granules are water-soluble and may be washed out during staining; thus they appear as empty areas in the cytoplasm Figure 8—2, B. A, Basophil. Lymphoblasts are difficult to distinguish morphologically in normal bone marrow.
Refer to Appendix Table A—1 for a comparison of myelocytes and lymphocytes. The criteria for diagnosis are listed in tables for ease of use, along with peripheral smear and bone marrow examples, and biopsy samples illustrating normal and abnormal results for both hematological and non-hematological diseases.
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