Laboratory Hematology Practice

By Bruce Davis

Expertly edited and endorsed by the International Society for Laboratory Hematology, this is the newest international textbook on all aspects of laboratory hematology. Covering both traditional and cutting-edge hematology laboratory technology this book emphasizes international recommendations for testing practices. Illustrative case studies on how technology can be used in patient diagnosis are included. Laboratory Hematology Practice is an invaluable resource for all those working in the field.

• Language: English
• Publisher: Wiley
• Release date: Jun 6, 2012
• ISBN: 9781444398571

Book preview

I: Cellular Analysis

1

Historical Perspective on Cellular Analysis

Elkin Simson

Mount Sinai School of Medicine, New York, NY, USA

Introduction

Cellular analysis in hematology has a fascinating history spanning more than three centuries. Blood cell analysis is noteworthy for a very high degree of technologic ingenuity, sometimes shown by rather unlikely people. Throughout, the inventions have been characterized by extremely careful observation, meticulous attention to detail, and the application of techniques advanced for their time. Throughout the centuries, analysis of cells, whether by the observational skills required for microscopy, the manual dexterity required for manual analytical techniques, or the advanced technical knowledge required to operate modern multiparameter analyzers, has always required a high degree of skill from the practitioners of the art and science.

Much of the material for this chapter has been obtained from the publications detailed in the reference section, within which the references to the original papers of the named individuals are detailed.

Microscopy

The origin of cellular analysis through the medium of microscopy is widely associated with Antonie van Leeuwenhoek (1632–1723) from Delft, in the Netherlands, although similar observations of blood cells were in fact first documented in 1668 in the personal documents of his fellow Dutchman Jan Swammerdam [1]. van Leeuwenhoek was however the first to publish his observations in a scientific journal [2].

van Leeuwenhoek (see Figure 1.1) was an unlikely scientist, a draper (fabric merchant), who came from a family of tradesmen, had no fortune, received no higher education or university degree, and knew no languages other than his native Dutch. This would have been enough to completely exclude him from the scientific community of his time; yet with skill, diligence, an endless curiosity, and an open mind free of the scientific dogma of his day, Leeuwenhoek succeeded in making some of the most important discoveries in the history of biology. His microscope consisted of a small lens that was essentially a bead of glass fixed to a brass plate, which was held close to the eye. Solid specimens were fixed to the point of an adjustable pin, while liquids were placed in tiny glass tubes. With this simple lens he discovered bacteria, amoebae, rotifers, and protozoa. He first observed the cells of blood in 1675, when he observed that his own blood was composed of small red globules, driven through a crystalline humidity of water. His estimates of red cell size were remarkably accurate [1,2].

Figure 1.1 Antonie van Leeuwenhoek.

(From http://commons.wikimedia.org/wiki/File:Anton_van_Leeuwenhoek.png.)

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The compound microscope, which consists of an eyepiece lens as well as an objective lens, was actually invented in 1590, well before Leeuwenhoek’s birth, by two Dutch eyeglass makers, Zaccharias Janssen and his son Hans Janssen. However, van Leeuwenhoek was able to achieve greater magnification and better resolution by skillful grinding of the lens of his simple microscope than by using the crude compound microscopes of his time. In the early 18th century compound microscopes were improved by the use of lenses that combined two types of glass, which were found to reduce the chromatic effect, the disturbing halos that result from differences in the refraction of light. A further significant advance in the mid-19th century was the development of achromatic microscopes, with objectives comprised of multiple lenses. The resultant images were sharp and well defined, with far better resolution than was possible with even the best simple microscope, and enabled further discoveries to be made. In 1842 Alfred Donné described platelets as the third cellular element in blood, and in 1875 Hayem introduced a method for counting them. Gulliver, in 1846, was able to differentiate between lymphocytes and granulocytes by size alone.

Paul Ehrlich, a man who contributed greatly in many spheres to the health of humanity, has been called the Father of Hematology, of Immunology, and of Chemotherapy. While still a medical student in 1877, he began to use aniline dyes to stain blood cells. He classified aniline dyes as acidic or basic and showed that one group of dyes preferentially stained the red blood corpuscles and eosinophil leukocyte granules, whereas the other group stained nuclei and lymphocyte cytoplasm. In 1879 he developed a neutral stain that could stain both groups simultaneously. With this stain, he documented the violet granules of the neutrophil leukocytes. He went on to describe in detail the appearance of lymphocytes, neutrophils, eosinophils, and basophils; and initiated the white cell differential in the form still used today.

In the 1860s Erb noted granules in the red blood cells of humans and animals that had been made anemic by venesection. These granules may have been reticulum or perhaps denatured hemoglobin similar to Heinz bodies. Ehrlich was probably the first investigator to describe the cells now regarded as reticulocytes, using methylene blue to stain the reticulum. In 1891 Smith described supravital-stained erythrocytes that contained reticulum in cattle with pernicious anemia. He felt that these cells represented not degenerative forms, but rather embryonic corpuscles, sent into the circulation before their time to make good the losses going on [3]. In the early 1930s Heilmeyer published descriptions of reticulocyte morphology at different stages of maturation, as well as the relative frequencies of these stages in the peripheral blood. Heilmeyer divided the reticulocytes into four groups, plus a group 0 for normoblasts that contained a nucleus as well as a dense perinuclear reticulum.

Manual Cell Analysis

The addition of quantitation to microscopic observation was a very important step in the analysis of blood cells. Manual methods of cell counting and cell characterization were all highly dependent on the quality of microscopes. Leeuwenhoek himself developed a method for counting the number of erythrocytes pulled into a glass capillary tube with graduation marks. In 1851 Karl Vierordt published a procedure for cell counting that required 3 hours or more to complete. He used a capillary pipette that was calibrated in diameter and length, the contents of which were expelled onto a flat slide where they were mixed with diluting and preserving fluid. The entire spread was then counted with the aid of a finely squared micrometer in the eyepiece of the microscope.

During the next 60 years many modifications of this basic procedure were introduced. In 1874 Malassez reported counting of white blood cells using an instrument called a hemocytometer, a shallow, graduated, rectangular chamber into which diluted blood was injected. To measure and mix the blood, pipettes were designed that sampled a fixed volume of blood and accurately diluted this before counting. A variety of hemocytometers and various diluting solutions were introduced. Red cells, white cells, and platelets were counted using this technique. The Neubauer hemocytometer, which consists of two chambers, each of which has finely ruled squares, has become the standard method for the performance of manual cell counts. This basic design is still employed when manual microscopic cell counts are done today.

Measurement of cell size was also initiated by van Leeuwenhoek. However, it was not until 1718 that Jurin accurately established the diameter of the human red cell. As with cell counting, measurement of cell size was performed visually until the 20th century. The magnified images of cells (usually flattened in a dried film of blood) were compared to a known dimension by calibrating the microscope.

Early in the 20th century, Wintrobe applied centrifugation techniques to whole blood, which enabled quantitation of the cellular fraction of the blood by measurement of the packed cell volume (PCV). Dividing this result by the RBC provided an indirect measurement of average red cell size. Although now largely replaced by automated methods that calculate the hematocrit (HCT) from the red blood cell count (RBC) and directly measure the size of red cells to obtain the mean cell volume (MCV; see Table 1.1), this manual centrifugation method is still, on occasions, used today.

Table 1.1 Red cell measurements and indices by manual and automated methods.

Hb, hemoglobin concentration; HCT, hematocrit; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; MCV, mean cell volume; PCV, packed cell volume; RBC, red blood cell count; RDW, red cell distribution width.

In 1949 Brecher described a method for quantitating reticulocytes by staining them with the supravital dye new methylene blue, then counting them using a microscope, with due note of Heilmeyer’s maturity classification described above, and calculating a ratio to the number of red cells. Manual reticulocyte methods are inaccurate and imprecise because of the difficulty in identifying the more mature reticulocytes and the low number of cells counted. They remain in widespread use, but are progressively being replaced by more reliable automated methods.

Hemoglobin

The earliest attempts to determine the concentration of hemoglobin in the blood included the visual matching of dilutions of whole blood to a liquid color reference by Gowers (1878), Hoppe-Seyler (1883), Sahli (1895), and Haldane (1901). The method of Sahli, in which the blood sample is mixed with hydrochloric acid to obtain acid hematin, is still used. In developing this technique it was found to be simpler and more quantitative to determine the color through the use of colorimeters and/or spectrophotometers; however, the spectral content of the various forms of hemoglobin precluded the choice of a good wavelength for measurement unless the hemoglobin was first converted to a single stable form. The determination of hemoglobin as cyanmethemoglobin or hemiglobincyanide (HiCN) was introduced by Stahe in 1920. The HiCN method has been studied extensively and was accepted by the International Council for Standardization in Haematology (ICSH) as the international reference method for hemoglobin analysis in 1964. Wintrobe used the measurement of hemoglobin and the PCV combined with the RBC to obtain red cell indices that indirectly measured the properties of the red blood cells, and in 1934 he published a classification of anemia based on the hemoglobin content and volume of red cells. The study of hemoglobin has been a prototype for the study of genotypes manifested into specific cellular and clinical phenotypes [4].

Single-Channel Analyzers

In the early 20th century, with advances in electronics and electro-optics, several attempts to simplify blood cell counting were made [5]. Moldavan, in 1934, described an apparatus in which a suspension of red blood cells was forced through a capillary glass tube on a microscope stage, each passing cell being registered and counted by a photoelectric apparatus attached to the ocular lens. He noted problems in standardizing the capillary tube, assuring proper focus, maintaining flow, and obtaining an appropriately sensitive photoelectric apparatus, and reported no further work himself. Around 1945 yet another instrument was described in which erythrocytes could be counted automatically by means of photoelectric spot-scanning of a thin layer of a diluted blood sample. This was an attempt to automate the manual counting chamber technique described above, in which the microscopist was replaced by a photomultiplier and an electronic counting unit, while the counting chamber was moved by a motor-driven system. This also failed.

Wallace Coulter’s discovery of an aperture impedance method, the Coulter principle, for counting and sizing cells, for which he obtained a patent in 1953, can be regarded as the origin of hematology automation. This principle made use of the lower conductivity of the erythrocytes compared with the diluting fluid. In Coulter’s instrument, blood cells suspended in an electrolyte solution were induced to flow through an electric field in a relatively short, small orifice drilled in a thin sapphire. The electric field in and surrounding this orifice was the sensing portion of the instrument, also called the aperture. Because of the small dimensions, diluted blood cells were readily detected and counted more or less individually without a high frequency of clogging. Cells could be sized simultaneously because the magnitude of the electrical impulse was found to be proportional to the cell volume. The first analyzer was the Model A (see Figure 1.2) and was followed by an alphabetically named series of single-channel analyzers, each of which contained successive improvements and additional features. An important feature of all these analyzers was that they aspirated, under mercury-manometer control, an accurate volume of blood. These analyzers were successfully used in thousands of laboratories worldwide and the Coulter principle provides the basis for most modern cell counters.

Figure 1.2 The Coulter counter model A.

(Image provided by Beckman Coulter.)

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Early Multiparameter Cell Analyzers

The first instrument to automate the performance of more than one cell count on a single sample was the SMA 4A-7A introduced by Technicon in 1965. In this instrument, each sample of blood was divided and diluted using continuous-flow technology that had been invented by Leonard Skeggs for chemistry analyzers. The cells in the sample were then counted individually with a photoelectric detector in two passes through a single, narrow flow cell, one without hemolysis of the red cells for the RBC, the other after hemolysis to obtain the white blood cell count (WBC). The hemoglobin content was determined in a separate and parallel channel after hemolysis of the red blood cells and conversion of the hemoglobin to cyanmethemoglobin. The instrument produced a seven-parameter complete blood count (CBC) on each specimen and operated at a throughput rate of 30 samples per hour. The instability of the dilution process using the continuous-flow method required the instrument to be recalibrated frequently, and it was not well accepted in laboratories.

The widespread use of a combined CBC analyzer was only achieved when Coulter introduced the Model S instrument in 1968. In this instrument, the sample was divided via a blood sampling valve and diluted into two glass reaction chambers, in one of which the red blood cells were hemolyzed. Samples from each chamber were passed through electrical counting apertures applying the Coulter principle to determine the RBC and the WBC. Hemoglobin was determined by optical absorption in the WBC reaction chamber without conversion to cyanmethemoglobin. The MCV was calculated from the average signal size in the red blood cell counting aperture. The HCT, mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were then calculated to produce a seven-parameter CBC. A maximum throughput rate of almost 100 samples per hour was possible by manually feeding samples into the analyzer, and the dilution stability achieved with the blood sampling valve and reaction chambers reduced the need for frequent recalibration. By the early 1970s the Model S had essentially revolutionized the hematology laboratory, consolidating routine testing into two workstations: the automated seven-parameter CBC and the remaining analysis. This second part of the analysis, typically called the leukocyte differential, still needed to be performed microscopically. Platelet counts were performed infrequently, using the microscopic manual method in which platelets were counted in a hemocytometer chamber, preferably using phase-contrast microscopy.

In 1970 Technicon introduced the Hemalog-8 instrument which added the platelet count to the seven-parameter automated CBC by counting platelets by photoelectric means in an additional parallel counting channel after hemolysis of the red blood cells. The PCV was obtained by automation of the centrifugal packing of red cells followed by a photoelectric scan that registered the cell/plasma interface. The system used an automated sampler, but because the continuous-flow dilution method was employed, periodic calibration was still required and it did not achieve widespread use.

In 1980 Coulter introduced the S Plus series, which added the platelet count to their automated CBC instrument. The platelet count was obtained simultaneously in the red blood cell counting aperture by discriminating between platelets and red blood cells on the basis of their signal size. A subsequent model, the S Plus II, added further parameters to the reported results of the automated CBC. These were the red cell distribution width (RDW), defined as the spread (coefficient of variation) of the red cell size distribution; the mean platelet volume (MPV), defined in analogy to the MCV of red cells by averaging the signal heights from the platelets; the lymphocyte percentage, which was defined by discriminating on the basis of signal size in the white cell counting aperture with the small cells labeled as lymphocytes; and the lymphocyte count, which was obtained by multiplying the lymphocyte percentage by the total WBC.

The next major advance in consolidation of cell counting was the introduction of the Coulter S Plus IV system in 1983. In this system white cells were classified into three categories: lymphocytes, monocytes (really mid-sized white cells), and granulocytes. In addition, the platelet distribution width (PDW) derived in analogy with the RDW was added. Three-part white cell differential counters remain in use to the present day, especially in smaller laboratories and physician offices.

Automation of the Leukocyte Differential Count

Intense activity was initiated during the 1970s in attempts to automate the leukocyte differential count. Two different technologic approaches were pursued. In one, a direct attempt was made to automate the microscopic procedure using pattern recognition and automated image analysis of a stained blood film. In the other, called the flow system, an attempt was made, using the general principles of automated cell counting, to differentiate the white cells after tagging them with specific stains.

One of the first achievements in automated image processing occurred in 1952. Papanicolaou, who had developed the Pap stain for cervical cytology, was also investigating an instrument intended to automatically screen cervical smears. He and Mellors discovered that cancer cells often emitted more fluorescence per unit area than normal cells, and they constructed a photoelectric scanner that automatically measured the nuclear fluorescence signals. This single parameter did not have enough discriminating power to screen for cervical cancer, so the instrument was not pursued further.

The next development occurred in the late 1950s and early 1960s. This was the Cytoanalyzer project, sponsored by the US National Cancer Institute (NCI), also for the purpose of screening cervical smears for the detection of abnormal cells. It was constructed to scan a slide and measure two parameters, nuclear size and nuclear density; it actually made several measurements of the features of any large dense objects in the field and classified these objects. However, because the logic was incapable of telling the difference between large dense areas that were the nuclei of cervical cells and those that were clumped leukocytes and other objects, it failed to work.

Prewitt and Mendelsohn, in 1966, constructed a research-oriented device, primarily used for the analysis of chromosomes. However, one of the first studies with the equipment involved the feasibility of blood cell classification. In the late 1960s and early 1970s, other image processing systems used for blood cell classification were developed by Ingram and Preston, Young, and Bacus. By 1973 algorithms to classify at least six major normal white cell categories, which included segmented and banded neutrophils, lymphocytes, monocytes, eosinophils, and basophils, were available, together with a means of evaluating performance results. These were incorporated into clinical laboratory instruments intended to automatically classify blood cells in a rapid, routine, and reliable fashion. One of the first instruments developed during this period was the Larc manufactured by Corning Glass and reported at the International Congress of Hematology in 1972. These were the first reports in the scientific literature of a routinely working automated white cell differential instrument in the hematology laboratory.

Subsequent to the development of the Larc system, additional commercial systems based on image analysis were developed and released by Geometric Data (Hematrak) in 1974, Coulter (Diff-3) in 1974, and Abbott (ADC 500) in 1978. In spite of the development of several instruments that produced a subclassification of the mature white cells, plus qualitative estimates of red blood cell morphology, and information regarding abnormal nucleated cells, these automated white cell differential instruments never attained widespread acceptance because the benefits obtained by their use did not compensate for their high cost.

The multiparameter cell counters achieved widespread popularity in routine hematology laboratories because they provided complete automation from incoming sample to reported results and completely eliminated the need to perform at least two microscopic cell counts as well as the hemoglobin and PCV determinations. On the other hand, the more expensive image processing instruments only automated the microscopic examination of the Wright-stained blood film and were relatively slow. Many blood films still had to be examined by microscopists because the automated systems had many imperfections, largely due to limitations in computer speed and the technology at the time. The preparation and staining of the blood film still had to be done manually (often to more exacting specifications than for visual observation). In addition, because most hematology laboratories at that time did not have laboratory information systems, the results had to be manually entered onto the reports from the cell counter.

Recently, there has been a resurgence of interest in image processing systems. Advances in computer hardware and software with the use of neural networking to enhance artificial intelligence capabilities have been incorporated into systems such as the Cellavision system (www.cellavision.se).

In parallel with the development of image processing systems, another approach to the automation of the leukocyte differential was made using enzyme cytochemistry as a means to differentiate the leukocytes, which were then counted and classified in an optical cell counting system. This approach was developed by Technicon in collaboration with researchers at Mount Sinai School of Medicine. Basic classification chemistries that had been developed and reported by Ornstein and Ansley in 1974 at Mount Sinai were automated using the continuous-flow methodology. The first automated differential system to result from this, the Hemalog-D, was released commercially in 1974. In this system the cells were classified in three parallel channels. In the first channel, myeloperoxidase-containing cells were stained by 4-chloro-1-naphthyl, and the resulting differentiation of cell color as well as size was then used to classify lymphocytes, neutrophils, and eosinophils. In the second channel, intracellular nonspecific esterase was used to specifically identify monocytes at a pH that favored their staining relative to other granulocytes. The third channel specifically classified basophils on the basis of their reaction with the Alcian blue stain that had been developed for the accurate counting of basophils visually in counting chambers. Therefore, the system completely automated the process, and in the Hemalog-D 10,000 cells were classified in each channel thereby achieving high reproducibility for the cell counts.

However, as with the automated differential instruments based on image analysis, the use of the flow-through differential systems was limited. Thus, even with the greater degree of automation achieved with these instruments, the benefits did not outweigh the costs. In many of the samples, microscopic evaluation was still required for red blood cell morphology and the identification of abnormal cells. Frequent recalibration was still necessary because of the continuous-flow technology. Technicon attempted to improve the benefits by combining the Hemalog-D and Hemalog-8 systems, using a combined blood sampler in parallel for the two systems. This combination produced the first prototype of the modern multiparameter cell counting system, which will be described later in this chapter.

Flow Cytometry

Although any measurement made on a cell or other object in a flowing stream is strictly speaking flow cytometry, by convention the term is now most frequently applied to those analyzers that perform optical and fluorescence measurements on cells that have been reacted with antibodies to various antigen markers on their cell surface.

As mentioned above, the first attempt to count cells automatically while in flow was reported by Moldavan in 1934. In 1953 Crosland-Taylor applied the laminar sheath-flow principle, which Reynolds had used in 1883 to study laminar flow and turbulence, to the design of a chamber for optical counting of red blood cells. An aqueous suspension of the cells was injected slowly into a faster flowing stream of fluid, which provided a laminar sheath that surrounded and aligned the particles. This approach overcame the problem of a narrow channel becoming blocked by large particles in the flow stream, and it also made precise centering of the narrow particle stream possible. Almost all flow cytometry instruments today make use of the sheath-flow principle described by Crosland-Taylor.

Kamentsky and colleagues in 1965 described the use of spectrophotometry to quantitate specific cellular constituents together with cell classification by a combination of multiple simultaneous measurements of different cellular features. They were able to display and analyze multiparameter flow cytometry data by means of a two-dimensional histogram. Subsequently, they reported a new cytometer capable of carrying out up to four simultaneous measurements per cell and were the first to record and analyze multiparameter data by an interfaced computer [6,7].

Fluorescent dyes provide important advantages over absorbing dyes as they greatly increase the sensitivity of detection. Fluorescent stains were first used by several different groups of researchers in 1967. Measurements of fluorescence at different wavelengths were combined with measurements of light absorption and light scatter.

An instrument to perform flow cytometry and sort viable cells by electrostatic means was developed in the late 1960s by Herzenberg et al. [7]. The flowing stream of cells was discharged into air at the nozzle of the flow cell, forming a very fine stream of droplets. Individual cells, identified before the stream broke up into droplets, were passively carried in the droplets. Droplets containing cells that met the sort criteria were charged at the moment of formation, and the charged droplets were sorted as they passed between constantly charged deflecting plates. This type of droplet separation was originally applied by Sweet in his invention of the ink-jet printer in 1965. Becton Dickinson introduced the commercial fluorescence-activated cell sorting (FACS) machines in the early 1970s and these systems, as well as those from other manufacturers, have been widely used to obtain pure populations of cells as identified by surface antigens.

The invention of hybridoma technology to produce monoclonal antibodies by Kohler and Milstein in 1975 was a major advance in the use of flow cytometry and cell sorting for research and clinical purposes. Each monoclonal antibody is highly specific for its target antigen and can readily be coupled to fluorescein, phycobiliproteins, and other fluorochromes. The use of monoclonal antibodies has enabled hundreds of target antigens present on or in cells to be defined.

The early flow cytometers, especially those with cell sorting capability, were large systems mainly used for research. In more recent times, smaller flow cytometers for clinical purposes have been developed, using only the analytical features of these instruments without cell sorting capability. They have been used primarily for lymphocyte subset quantitation and for leukemia immunophenotyping. In addition, several of the features of flow cytometers, such as fluorescence capability, have been added to multiparameter hematology analyzers.

Modern Multiparameter Cell Analyzers

The first modern multiparameter cell counting system was the Technicon H6000 introduced in 1981. In this system Technicon completed the combination described above by physically combining the cell counting technology that it had developed independently for the automated CBC (Hemalog-8) and the automated leukocyte differential (Hemalog-D) on a single platform. Redundant channels were eliminated and the H6000 produced a combined CBC plus five-part leukocyte differential at 60 samples per hour. The H6000 demonstrated the value of consolidating hematology testing into a single workstation; however, it was difficult to operate, required frequent periodic calibration and was not well accepted in laboratories.

The modern multiparameter cell counters represent further advancement of the multiparameter one-workstation approach. The first of the modern systems was the Technicon H*1 system, which was launched at the end of 1985. The continuous-flow approach was abandoned in favor of syringe pumps and this analyzer was reasonably well accepted by laboratories. The H*1 provided a combined CBC including platelets and white cell differential, with specific flags for any abnormal white cells detected that were not quantifiable by the analyzer, as well as red cell morphology flags. It was also able to quantitate hemoglobin within individual cells, a feature that remains unique to the Technicon/Bayer systems.

Other manufacturers introduced similar analyzers with similar features soon afterward. Coulter introduced the STKS analyzer in 1989 and the smaller MAXM analyzer in 1991. The Sysmex NE-8000 system was also introduced in 1989, with three separate channels to produce the leukocyte differential count. The Abbott CellDyn series of multiparameter analyzers, which used a multidetector optical cell counter with a polarized light source to perform the leukocyte differential count, was introduced in the late 1980s. Roche and Horiba ABX introduced multiparameter systems in the early 1990s.

Automation of the reticulocyte count began in 1989, when TOA Medical introduced a benchtop system, the Sysmex R-1000 Reticulocyte Analyzer specifically for counting reticulocytes. An upgraded R-series analyzer, the R-3000, was released 3 years later. Soon afterward, reticulocyte counting was added to the multiparameter analyzers, first by offline preparation with subsequent analysis on the system; and then by online fully automated analysis together with the CBC and differential.

More recent additions have included broadening of the menu of tests to add counting of nucleated red blood cells (NRBC) and immature granulocytes. An immunologic platelet count has also been added by Abbott. The features, capabilities, and use of the presently available multiparameter analyzers with continually extended cell differential counting will be described in the relevant chapters of this book.

References

1 Mohandas N, Gallagher PG. Red cell membrane: past, present, and future. Blood 2008; 112: 3939–3948.

2 Wintrobe MM. Blood, pure and eloquent: a story of discovery, of people, and of ideas. New York: McGraw-Hill Book Company; 1980.

3 Koepke JF, Koepke JA. Reticulocytes. Clin Lab Haematol 1986; 8: 169–179.

4 Schechter AN. Hemoglobin research and the origins of molecular medicine. Blood 2008; 112: 3927–3938.

5 Groner W, Simson E. History of Cell Counting. In: Groner W and Simson E. Practical Guide to Modern Hematology Analyzers. Chichester UK: John Wiley and Sons; 1995; 1–19.

6 Melamed MR, Mullaney PF. An Historical Review of the Development of Flow Cytometers and Sorters. In: Melamed MR, Mullaney PF, Mendelsohn ML. Flow Cytometry and Sorting. New York: John Wiley & Sons; 1979; 3–9.

7 Shapiro HM. Practical Flow Cytometry, 4th edn. Hoboken, NJ: John Wiley and Sons; 2003; 73–100.

2

Cellular Morphologic Analysis of Peripheral Blood

Powers Peterson¹, Sheila McNeill², and Gene Gulati³

¹ Quest Diagnostics Nichols Institute, Valencia, CA, USA

² Sentara Norfolk General Hospital, Norfolk, VA, USA

³ Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA, USA

Introduction

The history of qualitative cellular analysis originates from the 14th century in Italy with the introduction of spectacles to correct vision. In the late 16th and early 17th centuries opticians invented the compound microscope, most likely as a result of accidentally inverting a telescope. Because of its intrinsic absorption and refraction of visible light, the compound microscope allowed the visualization of small objects, although the magnifications possible were only ×20–30. In 1665 Robert Hooke, the English Father of Microscopy, published Micrographia. In examining the microscopic structure of cork, he analogized its organization to monks’ cells in a monastery. The word cell to describe the basic structure of biologic organisms remains to this day. Better known as the Father of Microscopy was the Delft scientist Antonie van Leeuwenhoek. He crafted microscopes that used double-convex lenses of high quality glass that he ground himself, which produced an optical magnification of slightly greater than ×200. With these he observed bacteria, muscle fibers, spermatozoa, lymphatics, and individual blood cells. van Leeuwenhoek first described red blood cells in 1674 and estimated their size as 1.1 times the value accepted as accurate today (approximately 7.5 µm).

The Prussian physician Rudolph Ludwig Karl Virchow is known as the Father of Pathology by virtue of his emphasis on the central role of the cell in biology. He stated in Die Cellularpathologie in 1858 that every cell originates from another existing cell like it. And he was perhaps the first physician to recognize leukemia, which became easier to describe with the advent of synthetic dyes that allowed reproducible staining of both microorganisms and blood cells.

Synthetic aniline dyes were the discovery of W. H. Perkin in 1853. In 1879 Paul R. Ehrlich, a German physician, expanded the use of these colorful dyes, classifying them as basic, acidic, or neutral. It was while trying to synthesize quinine that Ehrlich inadvertently made his discovery, which enabled reproducible staining of blood cells including the granules in white blood cells [1,2]. His acidic/basic dye combinations for blood cells became the basis for the Romanowsky [3], Giemsa [4], Wright [5], and May–Grünwald modifications [6]. Dimitri Leonidovich Romanowsky modified Ehrlich’s technique using an aqueous mixture of eosin Y and oxidized methylene blue [3]. Because the aqueous dye solutions were unstable, James Homer Wright introduced methanol as a solvent and advocated using methanol as a fixative prior to staining [5]. Gustav Giemsa standardized the dye solutions and added glycerol to increase solubility and stability [4]. Richard May and Ludwig Grünwald used saturated solutions to increase the intensity of the staining of the individual blood cells [6]. For general screening and surveillance purposes, Wright–Giemsa or May–Grünwald–Giemsa-stained films display unrivaled beauty and discrimination of form and color [7].

Every day throughout the world hematology laboratories use these stains to examine blood smears by light microscopy. Qualitative and quantitative descriptions of cellular changes are observed and recorded, and reports are duly issued. The percentage of complete blood count (CBC) specimens followed by a manual review, scan, or differential count ranges from less than 10% to more than 50% with an average of nearly 27% in laboratories the United States [8]. Is this labor-intensive, expensive activity really necessary? With the advent of optical and electronic cell counters for enumeration, immunophenotyping for proteomic characterization, immunocytochemistry for localization (nuclear, cytoplasmic, or membrane), and advanced techniques for detection and identification of molecular abnormalities, is light microscopic evaluation of the blood smear a clinically useful activity? The answer is unequivocally yes, because the blood smear remains a crucial diagnostic aid [9].

Atlases and articles abound with intimate details of the colors, shapes, and sizes of the cells that inhabit the peripheral blood, both in their normal forms, their variations, and in abnormal forms [10–16]. This chapter on cellular morphologic analysis will not recapitulate these extensive bodies of work. Instead the authors will concentrate on the peripheral blood smear as a unique diagnostic tool in the laboratory hematology armamentarium. This discussion will address technical topics as they relate most specifically to the blood smear: sample collection, pre-analytic variables that affect the quality of the blood smear, and staining artifacts. This will be followed by further discussions of blood cell examination, first with respect to the results generated by automated analyzers, the CBC, and then in terms of the examination of the peripheral blood smear. Subsequent discussion will focus on the diagnostic potential of the blood smear. There are limitations, but in the right clinical setting there is unquestionably clinical utility. What diagnoses are possible? Can a definitive diagnosis be rendered? Lastly, the authors have constructed figures to illustrate those conditions, clinical and artifactual, in which definitive findings are present on peripheral blood smears.

Sample Collection

A significant variety of clinical information can be obtained from a well-made, well-stained, and thoughtfully analyzed blood smear [9,17,18]. According to Jandl more information can be gained from examining the blood smear than from any other single hematologic procedure [7]. Proper sample collection, processing, and staining allow the laboratory professional and/or clinical physician to identify spurious results, reach a diagnosis, and suggest further testing if warranted.

Blood sample collection must follow standard precautions. Proper patient identification is the critical first step in the collection process. The method of blood collection is based on the patient’s age, physical condition, and the volume of blood needed. Venipuncture utilizing an evacuated tube system is the most frequent sampling technique. Use of a syringe or winged infusion set is generally reserved for small or fragile veins. Skin punctures are performed primarily on newborns and pediatric or geriatric patients.

For a venipuncture, the needle must be engineered with sharps injury protection in mind and the needle holder is for single use only. The preferred venipuncture site is a vein in the antecubital fossa, wrist, or hand. A tourniquet, usually a latex band, is applied 7–10 cm (3–4 inches) above the draw site and secured for no more than 1 minute. The selected site should be cleaned with 70% isopropyl alcohol and allowed to air dry. The puncture should be made with the needle bevel-up at approximately a 30° angle. Following the puncture, a tube is applied to the needle inside the needle holder. The order of draw is prescribed and the first draw should be for blood cultures if required, in which case the skin cleansing should be performed with betadine. This is followed by: sodium citrate, plain, gel separator, heparin, ethylene diamine tetraacetic acid (EDTA), acid citrate dextrose, and oxalate or fluoride [19]. Each tube is allowed to fill, and as it is pulled from the needle holder, it must be mixed by inversion according to the manufacturer’s recommendations. Tubes must be filled to within 10% of the recommended volume. When collection is accomplished and the needle is removed from the draw site, pressure is applied to the site. All tubes should then be labeled immediately with the appropriate patient information, date and time of draw, and the identification of the person obtaining the specimen(s). The needle and its holder should be disposed of in designated sharps biohazard containers only.

Blood collection with a syringe differs from that of a venipuncture with needle holder in two aspects. First, the pressure applied to the barrel provides the vacuum to fill the syringe with blood. Second, a blood transfer device that resembles a needle holder with an attachment to connect to a syringe is then attached to the syringe to aliquot the blood, which is done by attaching tubes to the needle inside of the holder. This is the currently approved method in the United States for blood transfer from syringe to laboratory tubes.

A winged infusion set has a smaller needle connected to tubing that can then be connected to a needle holder. Because the tubing contains air, a sodium citrate tube must not be drawn first. If a sodium citrate tube is the only draw, then a blank tube must be utilized first to remove the air from the collection set.

Skin punctures are performed primarily on newborns and pediatric or geriatric patients. If indicated, capillary specimens can also be collected directly into Unopettes or lavender-capped microcontainers for cell counting. Direct blood smears may be made from the microcontainer or from the puncture site after the first drop of blood is wiped away. When a skin puncture is performed, the first tube filled is the EDTA or other anticoagulant-containing microcollection tube. This will ensure more accurate hematologic results.

The vacuum tubes for blood collection and microcollection tubes have colored caps that follow a universal coding system among manufacturers. For hematology specimens EDTA is the recommended anticoagulant [19–21]. Dipotassium (K2) EDTA powder is sprayed onto the wall of plastic lavender-capped tubes. The use of plastic tubes mitigates many of the issues regarding the safety of glass tubes and the potential risks associated with their breakage. Wherever possible, glass tubes are being phased out of use [22].

Pre-Analytic Variables

Transport of specimens to the laboratory should occur as soon as possible. They should remain at room temperature, with the one exception of specimens obtained from patients with severe cryoglobulinemia or cold agglutinins. These specimens may need to be transported at as near as possible to body temperature. Analysis by an automated hematology instrument should ideally be performed within 6 hours for EDTA-evacuated tubes and within 4 hours for microcollection tubes [20,23]. The integrity of a specimen, which includes proper labeling and correct fill volume, should be verified at analysis. Any specimen with visible clots must be rejected. Adequate mixing ensures accurate cell counts on a hematology analyzer. As delineated in Table 2.1, there are also patient conditions that can affect a single CBC. Results of an isolated CBC may not be a true indicator of patient status without the patient’s clinical history. An experience of one of the authors serves as an example: a patient presented with a WBC of 10 × 10⁹/L, which was within the reference range. However, this patient’s normal WBC was 3 × 10⁹/L and the patient in fact had an acute infection that required antibiotics.

Table 2.1 Variables that may affect complete blood count (CBC) results.

RBC, red blood cell count; WBC, white blood cell count.

Peripheral Blood Smears

Wedge pull smears are made after the tube has been mixed either by inversion at least 20 times or by placing on a tube rocker. A small drop of blood is placed at one end of a clean glass slide with a microhematocrit tube or a specific transfer device. The spreader slide touches the slide at a 30° angle and is placed slightly in front of the drop of blood. It is pulled back until contact with the blood is made and then pushed forward in one continuous motion. This results in a smear with one edge that is rectangular and no visible blood droplet. The rounded end is feathered and the smear appears smooth with no streaks or ridges. All slides are labeled with patient information.

After being air-dried, the slides can be stained manually or on an automated slide stainer [23]. For manual staining, the slides are placed on a staining rack over a sink, and stain, buffer, and rinse are applied in a timed fashion. There are two types of automated stainer. The first is a dip stainer that holds the slides vertically either in a basket or in individual cassettes and robotically moves them through the stain, buffer, and rinse processes. The second has two conveyer spirals and each slide is pushed along a platen and individually stained. With the robotic systems common in high-volume laboratories, a tube of blood may be directed to a robotic slide maker/stainer. There the blood is mixed, then a wedge pull slide is made, which is labeled and stained.

Artifacts on Peripheral Blood Smears

Regardless of whether a blood smear is manually or automatically generated, artifacts may be present [24]. The first step to correcting the problem of an artifact is to recognize it as such, whether it is due to an abnormality in the patient, for example an elevated hemoglobin level, or to a technical problem, for example stain precipitation. Table 2.2 lists the most common reasons for artifactual changes on a blood smear and makes recommendations for correction of the problem. Some artifacts cannot be corrected except by obtaining a new specimen. Specimens that have been exposed to high temperatures or that have sat around for more than 6 hours show irreversible changes [25].

Table 2.2 Artifacts that can be present on peripheral blood smears.

Blood Cell Examination: the Complete Blood Count

Blood cell examination is usually performed in a sequential manner, beginning with analysis by an automated instrument. Samples are selected for further analysis if quantitative or qualitative abnormalities are found [8,26–34]. Quantitative abnormalities include aberrant values for cell counts or cell size for the instrument-generated leukocyte differential. Qualitative abnormalities include alert flags that may indicate the possibility of inaccurate results or the presence of abnormal cell types. Qualitative abnormalities vary in clinical importance. Some reflect expected variations in clinical circumstances; others indicate conditions that warrant attention.

A related issue is that of false-positive and false-negative results from automated analyzers [7,27–31,35,36]. Falsely abnormal results suggested by automated analyzers can be identified with careful observation of the blood smear. Table 2.1 lists the potentially overlooked but unequivocally important variables that can affect results generated by an automated analyzer. Equally important, a normal result from an automated analyzer does not exclude the possibility of an inherited or acquired hematologic or other disorder, examples of which are listed in Table 2.3. Examination of the blood smear may clarify whether the numerical result from the analyzer is spurious (Figure 2.1) or real (Figures 2.2 and 2.3). An example of a condition that can cause a spurious result is shown in Figure 2.4. (Note: Figures 2.4–2.13 are all images from blood smears stained with either Wright or Wright–Giemsa stains.)

Table 2.3 Conditions in which the complete blood count (CBC) may be unremarkable but examination of the blood film will suggest or confirm a disorder.

Modified from Ryan DH [44], with permission from McGraw-Hill.

Figure 2.1 Causes of spurious complete blood count (CBC) results from automated analyzers. These conditions can be diagnosed on examination of a peripheral smear. Some conditions causing erroneous (spurious) results are artifactual; others reflect biologic variations or abnormalities. Some clinical conditions, such as hyperlipidemic states and extreme leukocytoses, may give spurious results on particular analyzers.

* instrument dependent; NRBC, nucleated red blood cell; WBC, white blood cell count.

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Figure 2.2 Medical disorders or conditions that cause increased or decreased cell counts from automated analyzers. These abnormalities can be diagnosed on examination of the peripheral blood smear.

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia; CTCL, cutaneous T-cell lymphoma; ET, essential thrombocythemia; G6PD, glucose-6-phosphate dehydrogenase; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; Hb, hemoglobin; LGL, large granular lymphocytosis; MPN, myeloproliferative neoplasm; PV, polycythemia vera.

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Figure 2.3 Medical disorders or conditions that cause morphologic cellular abnormalities that can be diagnosed on a peripheral blood smear. These disorders may be genetic (hereditary) or acquired.

* extra-erythrocytic organisms; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; B. recurrentis, Borrelia recurrentis; CLL, chronic lymphocytic leukemia; CML, chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia; CTCL, cutaneous T-cell lymphoma; ET, essential thrombocythemia; G6PD, glucose-6-phosphate dehydrogenase; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; Hb, hemoglobin; HPPK, hereditary pyropoikilocytosis; LGL, large granular lymphocyte; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; PMF, primary myelofibrosis; PV, polycythemia vera.

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Figure 2.4 Platelet satellitosis.

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Figure 2.5 Wuchereria bancrofti.

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Figure 2.6 β-thalassemia major (β⁰).

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Figure 2.7 Glucose-6-phosphate deficiency—hemolysis with characteristic bite cells.

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Figure 2.8 Chediak–Higashi syndrome.

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Figure 2.9 AML, M3—example of a faggot cell.

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Figure 2.10 Megaloblastic anemia—examples of a six-lobed polymorphonuclear leukocyte and a Howell–Jolly body.

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Figure 2.11 Babesia microti—examples of multiply infected red blood cells, including one with a tetrad form.

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Figure 2.12 Histoplasma capsulatum infection.

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Figure 2.13 Bordetella pertussis infection.

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All laboratories should have a protocol for the examination of a laboratory-initiated blood smear [9,38]. A comprehensive set of guidelines for laboratories worldwide has been developed and validated by an international consensus group for hematology review and published by the International Society for Laboratory Hematology (ISLH) [26; www.islh.org]. The suggested criteria, or rules, as they relate to generating a slide for review are shown in Table 2.4. Ideally, a laboratory’s protocols take into account clinical data such as the age and sex of the patient, analyzer-generated results, and laboratory organization and resources. Hierarchical protocols vary among testing sites and are structured to reflect the level of training and experience of testing personnel, the sophistication of the automated analyzers, and the incidence of variations or abnormalities in the population being tested [39]. For each laboratory, the written procedures delineate which quantitative and/or qualitative abnormalities mandate a microscopic review of the blood smear and by whom this should be performed [26,40–43]. When abnormalities are unfamiliar, rare, or potentially significant for diagnostic and/or therapeutic reasons, a physician or laboratory specialist may need to review the blood smear. Optimally, integration of clinical information with the numerical and/or morphologic abnormalities improves the quality of laboratory results and enhances patient care [37,44].

Table 2.4 Reasons for slide review based on results from an automated analyzer.

#, absolute number.

Extensively modified, but consistent with International Society for Laboratory Hematology (ISLH)-endorsed international consensus recommendations [26].

MCV, mean cell volume; NRBC, nucleated red blood cell; RDW, red cell distribution width; WBC, white blood cell count.

Blood Cell Examination

An initial scan of a stained blood smear at low power will allow assessment of the quality of the slide, distribution of the cells, and quality of the stain [10,18,23,37,45,46]. A poorly made slide with ridges in the smear will yield inaccurate results and a new slide must be made. Stain precipitate deposited on the slide indicates the need to troubleshoot the staining process. The stain tube lines will need to be cleaned with methanol and the rinse lines checked for fluid before a new slide is stained. A large number of smudge cells with nuclear remnants of lymphocytes on a smear may indicate the presence of fragile cells. These can be reduced by adding one drop of 22% albumin to five drops of blood and making a new blood smear from this mixture. The presence of red blood cells that appear too pink or blue on an initial scan indicates the need to verify the pH of the stain and/or the quantity of the buffer. Increased proteins can also cause the slide to have a bluish-purple appearance.

Review of the blood smear can result in a more rapid and accurate diagnosis of a variety of hematologic and other disorders [7,44,47–49]. These include malignancy as well as infectious, congenital, and acquired disorders. Specific findings in the peripheral blood smear can tailor or truncate further testing. For example, the detection of red cell agglutination in a patient with a previously undiagnosed anemia might suggest that further studies should be done for an autoimmune hemolytic process. If the red blood cell finding were rouleaux instead, further studies for a plasma cell dyscrasia, such as myeloma, would be indicated. The finding of fragmented red blood cells could imply a microangiopathic process, a severe megaloblastic anemia, or a more benign disorder.

Peripheral blood smear findings may yield an unequivocal diagnosis that dictates the therapeutic option. For example, the classification of a parasitemia as Plasmodium infection instead of Babesia infection, or Wuchereria bancrofti (Figure 2.5) instead of Loa loa or Mansonella perstans infection would permit appropriate therapy to be initiated. In other cases, a diagnosis can be suggested but additional confirmatory studies are warranted. For example, the presence of nucleated red blood cells (NRBCs) in an adult could indicate any of the following: recent significant blood loss; a hemolytic process, either congenital such as thalassemia (Figure 2.6) or acquired such as severe thermal injury; myelodysplasia; or acute erythroid leukemia. Basophilic stippling of red cells indicates disordered erythropoiesis, which could also indicate any of the medical conditions listed for NRBCs. In a child, however, the possibility of lead poisoning should also be considered. Table 2.5 summarizes the possible diagnostic utilities of review of the blood smear.

Table 2.5 Reasons for review of a blood smear.

Modified from Peterson P et al. [51], with permission from Carden Jennings Publishing.

DIC, disseminated intravascular coagulation; G6PD, glucose-6-phosphate dehydrogenase; TTP, thrombotic thrombocytopenic purpura.

Limitations of the Blood Smear

If Table 2.5 represents the reasons to review the blood smear, then what are the limitations of peripheral blood smear review? The major limitation is that identifying an abnormality is not necessarily equivalent to making a diagnosis. Table 2.6 elucidates on this concept. For example, two of the categories of hematologic malignancies listed as qualitative (leukemias and stem cell disorders) both require additional and far more sophisticated laboratory studies for an unequivocal and accurate diagnosis. It could be hazardous to classify an acute leukemia on the basis of a peripheral blood smear alone. In the category of both qualitative and quantitative abnormalities, the limitation of evaluating cytopenias is identical to that stated above for malignancies and stem cell disorders (myeloma, paroxysmal nocturnal hemoglobinuria). The same applies to the hereditary hemolytic disorders. Although there are hemoglobinopathies in which the peripheral blood smear findings are nearly diagnostic, such as hemoglobin SS [50] and hemoglobin CC, any such finding necessitates further confirmatory laboratory study and family studies. A similar logic follows for potential red cell enzyme (Figure 2.7) and membrane defects detected on smear review.

Table 2.6 Limitations of peripheral smear review.

RBC, red blood cell count; WBC, white blood cell count.

Clinical Utility of the Blood Smear

With these limitations in mind, review of the blood smear is an essential medical step in suggesting or even making a diagnosis. Table 2.7 presents a medically, results-oriented way of summarizing the rules listed in Table 2.4. An intelligent and thorough examination of the blood smear will be sufficient to correctly diagnose some disorders. Inherited leukocyte and platelet disorders