The ABC of CBC Interpretation of Complete Blood Count and Histograms
The ABC of CBC Interpretation of Complete Blood Count and Histograms
Second Edition
DP Lokwani MD
Former Founder Vice-Chancellor Madhya Pradesh Medical Sciences University Jabalpur, Madhya Pradesh, India
Professor and Head Department of Pathology Netaji Subhash Chandra Bose (NSCB) Medical College Jabalpur, Madhya Pradesh, India
Former Honorary Dean (Academics) Diplomate National Board Courses Jabalpur Hospital and Research Center Jabalpur, Madhya Pradesh, India
Sunit Lokwani MD DM
Consultant Hematologist and Medical Oncologist Shalby Group of Hospitals Jabalpur, Madhya Pradesh, India
Foreword
MB Agarwal
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The ABC of CBC: Interpretation of Complete Blood Count and Histograms
First Edition: 2013
Second Edition: 2022
9789390595983
Printed at
Babaji—My Spiritual Master
My Parents—My Blessings
My Teachers—My Strength
My Students—My Treasure
My Patients—My Experience
Foreword
Hematology has advanced by leaps and bounds during the last two decades. Sophisticated laboratory investigations have moved from research to clinical laboratories. This is an era of polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), flow cytometry, etc. One may feel that trying to read a book dedicated to complete blood count (CBC), may not be worth the time spent. However, we teach our students that in clinical practice, history and examination of the patient are of more importance than various investigations ordered costing thousands of rupees. Similarly, a well-executed “CBC” followed by its proper interpretation has its worth in gold and a shrewd clinician can make tremendous use of this simple and cheap test for diagnosing hematological or even non-hematological disorders.
All of us order CBC as the first and unavoidable test in our clinical practice. A student also talks of CBC as a routine test without giving it any importance. To write a book on such a test is bringing back some respect to the test that it deserves.
DP Lokwani has done a laudable job in writing this book on “CBC” and histograms in the era of sophisticated laboratory techniques. I have been told that he has utilized data collected over thousands of CBCs and histograms to prepare this book and that makes the book even more important. There is no match to the knowledge generated out of the experience and this is what he has done. The book has been written based on material collected over seven long years of his huge practice and hence the information provided is useful not only to students but also to the practicing clinicians and teachers in the field of hematology, pathology, and allied branches.
I had the opportunity of going through the book which also has 30 case illustrations. This makes the discussion even more lively and practical. I have no hesitation in recommending the book to be on the shelf of everyone even remotely connected with CBC.
MB Agarwal MD MNAMS
Head
Department of Hematology
Bombay Hospital Institute of Medical Sciences
Mumbai, Maharashtra, India
Book Review
Despite the enormous advances made in immunophenotyping and cytogenetics and molecular aspects of hematology, the analysis of the composition of peripheral blood and the observation of the peripheral blood smear at the microscope remains fundamental in the diagnosis of hematological disorders, as well as in many other areas of clinical medicine. The complete blood count (CBC), in effect, has evolved during the last decades from a short-list of numerical results to a real-time representation of the functional status of hematopoiesis. New automated instruments have immensely improved the speed and reproducibility of quantitative measurement of the EDTA-anticoagulated blood. Original cellular parameters, in addition to the classical CBC and WBC differential count, have been devised and made available to the laboratory operators, such as RDW, IRF, CHr, Ret-He. Flags for abnormal cells are important to identify samples that need a microscope review, and their sensitivity and specificity are continuously improving with a reduction in the rates of false-positive and false-negative results. Such instruments, including the morphology of cell distribution in histograms and scattergrams, must be interpreted by the trained and skilled human eye and brain so that the full spectrum of diagnostic information present in such a simple analytical report is fully exploited. Thus, the study of the new methods is a necessary complement to practice for those who are approaching the laboratory hematology of the year 2000. Similarly, the knowledge of hematological pathologies and cellular aberrations in the different diseases remains a cornerstone for the interpretation.
The ABC of CBC, wisely conceived, scientifically devised, and skillfully written by DP Lokwani, represents an excellent and authoritative reference tool to answer the need for knowledge in the area of laboratory hematology.
Besides a clear and comprehensive text, the book is full of microscope cell images, diagrams, histograms, and scattergrams. It indicates to the reader the best approach to fully appreciate and exploit all the new information and the advanced diagnostic features of the current automated technologies for CBC. Throughout the pages, it is easy to appreciate the value of the wide spectrum of clinical information present in an instrumental report. In the different chapters, we find technical descriptions of diagnostic methods, often in a historical perspective (for instance, see page 7 and chapter 6), as well as a description of both the basic and the new hematological measurements. These include some valuable time-honored practical information, such as the “rule of 3” (page 12). The pages are also rich in colorful drawings, visually illustrating some otherwise abstract concepts so that the rigorous scientific approach does not impede the Author's lively creativity and artistic talent. the use of images is very important to catch the imagination, the interest, and the concentration of students and readers; a representative and vivid figure will never be forgotten. Updated scientific information also covers very specific areas, with significant practical impacts, such as the possibility of detection of malaria parasites using automated blood cell count (page 31) or the importance of osmotic changes in red cells due to hyperglycemia (page 105). Chapter 6 describes the interwoven roles of automated and manual methods, while chapter 7 gives detailed and original clues for the interpretation of cell distribution in the instrument histograms. Cell distributions in a large number of disorders are also shown and explained, from aplastic anemia to HBH disease, from iron deficiency to myelofibrosis. Chapter 10, in which the author presents 30 well-documented clinical cases selected to represent a wide range of clinical conditions is among the most valuable features of this textbook. Finally, the author does not forget that perfect technical quality of testing represents an unavoidable premise to any diagnostic observation; the last chapter thus reminds the important rules of quality control, that all laboratory operators must know and use with great attention.
“The ABC of CBC” indicates to us how correct and informed use of the automated blood cell count can represent a bridge between the traditional laboratory diagnosis of blood disorders and the latest diagnostic technologies. Discoveries and capabilities have not canceled or replaced the classical methods, but have increased their value. Even in the era of molecular medicine textbooks like this still play a central and unique role in the training of the new generation of blood professionals in laboratory hematology.
Prof. Gina Zini
Director of Blood Bank
Director of UNICATT Cord Blood Bank
Fondazione Policlinico Universitario A. Gemelli IRCCS - Roma
Università Cattolica del Sacro Cuore, Rome
Preface to the Second Edition
The second edition, a step forward from the first edition, has been derived from the marked increase in documented innovations over the last few years and their applicability in diagnostic improvisation.
Increasingly enough, medical practitioners have recognized the use of these improved techniques and equipment. I have kept the essential backbone of the informatics; the story is the same as in the previous edition.
All the old chapters have been overhauled, most of them significantly restructured, updated, and extended. There are many new sections within the updates chapter covering relevant recent advances and applications in clinical practice.
As the test menu in the clinical laboratory is complex and as computer's increasing ability to generate and interpret newer and newer parameters within the same sample of CBC is getting incorporated in the cell counter technology, it becomes a call of the times to update the commonly referred elaborate document ABC of CBC for the better understanding of this very basic and most demanded investigation.
Updates in this ever-evolving subject have been happening by leaps and bounds. It seemed a foolhardy mission for me to cope up with the ever-changing dynamism of the subject of CBC, with the diversity of philosophies and their products of cell counters of various brands, this homogenous take would not have been possible without the help of Dr. Sunit Lokwani, who made this exercise more workable in the available time as well as in the scientific frame.
Apart from many relevant changes and additions made to each chapter, the most liked chapter of this book “Interpretation of Histograms” has been enriched with RBC cytograms and WBC scatterograms in various disease conditions with their interpretation and practical application in diagnostic medicine.
DP Lokwani
Preface to the First Edition
Diagnosis is not the end but the beginning of practice—Martin H Fischer
The complete blood count (CBC) is one of the most frequently ordered and routinely done investigations in the hematology laboratory. It is a Pandora's Box of information, which aids in the diagnosis of a multitude of diseases and disorders in the human body. This simple investigation, aptly labeled as the “meat and potatoes of hematology”, is economical, the time-honoring, minimally invasive, and exorbitantly informative.
This book is a navigation guide for students and practitioners alike related to pathology and clinical subjects, as nearly all body ailments have a direct or indirect influence on the blood picture. Hematology is all about relationships, like the relationships of the bone marrow to the systemic circulation, the plasma environment to the red cell lifespan, and hemoglobin to the red cell, so the book and you, the students and practitioners, are a vital part of this relationship.
With the changing dynamics of technology, the last decade has brought forth a radical change in hematology. The wheels of hematology have moved from predominantly manual practice to highly advanced automation. The Coulter principle and Coulter counter was a landmark in the field of hematology, and the prolific Coulter's revolutionized laboratory procedures changed the face of medicine by turning hematology guesswork into accuracy. What is needed for the novice practitioner is a way to approach interpreting the visual automated data. This skill is neither necessarily practiced at university programs, nor much of the literature is available in the racks of medical libraries dedicated to the subject of CBC in this era of automation.
The book aims to help the student bridge the gap between the classroom and clinical practice; introducing the automation principles in hematology along with interpretation and deciphering of various parameters like red cell distribution width (RDW) and histograms which carry a lot of encrypted diagnostic information because eyes can see only those things which brain knows. Although the data of the automated blood count alone will not guarantee specific diagnosis in every case, yet does every disease have a single pattern of abnormality. Rather these values should be used to narrow the differential diagnosis as much as possible, so that more expensive and time requiring, a confirmatory battery of expensive investigations like vitamin B12, folic acid, serum iron, total iron-binding capacity (TIBC) estimations, or Hb electrophoresis, etc., is done only selectively and invasive procedures like bone marrow aspiration and biopsy can be avoided, by proper evaluation of CBC.
The approach of the book is as simple, straightforward, and user-friendly as possible. I hope that clinical residents and consultants will find the book useful and also the text will travel with you, as you continue your career in the laboratory profession and the information will motivate you arousing your intellectual curiosity.
Attempt is virgin, mistakes are mandatory, corrections and suggestions are solicited.
DP Lokwani
Acknowledgments
A long time back, I had this dream, a vision of presenting a book on Hematology to the members of my fraternity.
And here I am with my pristine venture, 2nd edition of The ABC of CBC.
First and foremost, I thank Babaji (My Spiritual Master), for giving me the ability, skill, and courage to complete this arduous task and fulfill this long-awaited dream.
Most humbly I am indebted and grateful to my mother, Mrs Heerdevi, for her incessant prayers and blessings and to my father, Mr Kishinchand Lokwani, for making me what I am today and instilling in me his virtues.
I am immensely grateful to my siblings, Shobha, Roopa, Vasu, Chandar and Pushpa, for having staunch faith in me and my capabilities.
I acknowledge my gratitude to all my teachers, both formal and informal, who have influenced my professional standing the most with a special mention to Dr PL Tandon and Dr BC Chhaparwal.
It is gratifying to be able to acknowledge at this platform my immeasurable gratitude and admiration for Dr MB Agarwal, who has been my role model and a perpetual source of inspiration.
I will fail in my duty if I don't especially mention the hardwork put in by beloved Shweta to shape the 2nd edition.
My professional growth has been nurtured by my supportive wife, Dr Lakshmi Lokwani, who made it possible for me to spend endless uninterrupted evenings at my desk, and encouraging me all the way.
I am immensely grateful to my beloved friend and my source of inspiration Dr Rajesh B Dhirawani, an eminent maxillofacial surgeon, who has guided the sails of my life towards a prosperous shore.
A very special mention to Dr Pushpraj Singh Baghel, my student, for his untiring efforts and dedication; without his support, this mammoth task would not have been completed in time. He has spent with me endless strenuous hours in the shaping and framing of the book. He has been a cardinal buttress in the foundation of the book. I consider myself extremely fortunate to have such a prized student and also thank my student Dr Apoorva.
I sincerely acknowledge the efforts and elementary research on this subject by Dr Bessman, who systematically addressed the use of histograms and dedicate to him, with deference, humility, and respect.
A special thanks to my workplace, Jabalpur Hospital and Research Center, Jabalpur, Madhya Pradesh, India, and all its consultants and patients and my colleagues at Netaji Subhash Chandra Bose (NSCB) Medical College, Jabalpur, Madhya Pradesh, India, and Mahatma Gandhi Memorial (MGM) Medical College, Indore, Madhya Pradesh, India.
Most importantly, my beloved children, Anita, Manisha, Hitesh, Kavneet, and my dearest granddaughter Kavya, for being my source of energy. I will fail in my duty if I don't especially mention the hard work and skilled efforts put in by my daughter-in-law Dr Shweta Lokwani to shape the 2nd edition.
It will be lapse on my part if I don't appreciate the cooperation and contribution of cell counter research and manufacturing giants Siemens, Mindray, Abbott, Sysmex, and others.
Gratitude towards all the students, practicing doctors, and all readers who have taught me so much about laboratory medicine and inspired me to bring forward this second edition.
Sincere thanks to Prof Gina Zini of Rome for her very sincere review with expert guidance.
I am extremely grateful to M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, for giving me this opportunity to dissipate my work to the people of my fraternity.
Glossary of Terms Employed in Automation and Hematology
- Anisochromia: Variation of hemoglobinization of cells.
- Anisopoikilocytosis: Anisocytosis is variation of cell size more than normal. Poikilocytosis is variation of cell shape. In practice, the two terms are used as a single phrase “anisopoikilocytosis” means variation of cell size and shape.
- Aperture: A small opening in a device by which blood cells may be drawn into an analyzer for counting.
- Backlighting: An alert mechanism to visualize a potential problem or an error. The instrument can highlight a parameter that falls outside the reference interval.
- Cluster Analysis: A type of analysis that is based upon the instrument's ability to cluster different cell populations together based upon size, staining characteristics, absorption characteristics and other parameters. The analysis of abnormal cell populations is accomplished.
- Coincidence: When two or more cells pass through an aperture at the same time and are counted as a single cell.
- Continuous Flow Analysis: Analysis of cells that flow past a laser beam and is based upon the cellular characteristics.
- Contour Grating: A means of analysis where information is plotted on an x-, y-, and z-axis. The result is a three-dimensional plot that can separate subpopulations of cells.
- Coulter Principle: Sizing and counting cells by detecting and measuring changes in electrical resistance when a cell passes through a small aperture.
- Cytometry: Measurement of the cell can be visual or automated.
- Diffraction: The ability of a light beam to break up into its component parts as it bends across the surface of a cell. Light is bent to project at a new angle.
- Dimorphic: Two populations of cells in a single sample of blood.
- EDTA: Anticoagulant commonly used for hematologic studies.
- Electrical Impedance: Cell counting principle is dependent upon the detection and measurement of changes in the electrical resistance produced by cells in a conduction solution as they move through an aperture.
- Erythropoiesis: The process of RBC production in the bone marrow.
- Flow Cell: A structure made of quartz. Quartz is transparent and does not bend the light that passes through it. It also allows ultraviolet light to pass through. Glass does not have these two properties. The counting of the cells and their evaluation occurs in the flow cell.
- Forward Angle Light Scatter: Light from a laser source is scattered in a forward direction (at a zero degree angle) when it strikes a cell or particle. The larger the object, then there is more forward light scatter.
- Forward High Angle Light Scatter: Similar to forward angle light scatter, except the forward direction is with a 5 to15° variation.
- Forward Low Angle Light Scatter: Similar to forward angle light scatter, except the forward direction is with a 2 to 3 degree variation.
- Hemoglobinopathy: Amino acid abnormality of the globin component of the hemoglobin molecule.
- Hemolysis: Accelerated destruction of the “short-lived” RBCs. It may be due to intrinsic (defect of the red cell itself) or extrinsic (destructive environment) causes.
- Histogram: A visualization graph that can be created based upon cell size and/or cell number and can indicate frequency of distribution.
- Hydrodynamic Focusing: The ability of an analyzer to create a gradient differential and cause cells and/or particles to flow in a single column in which a central column of the sample fluid flows inside a column of sheath fluid. The laminar flow physics principles cause the sample stream to flow in a narrow lumen and at a rate faster than the sheath fluid. This narrowing phenomenon allows the cells to separate and align into a single file for passage through the sensory zone of the flow cell.
- Hypoproliferative: Quantitative decreased blood cell formation, also known as hypoplastic, the extreme of which is aplastic.
- Isovolumetric Sphering: A technique in which cells are placed in a buffered diluent that results in each cell becoming spherical, without altering the volume of the cell.
- Laminar Flow: A mathematical and physical term that describes the flow properties of fluids as they move through a tubular system. In laminar flow, particles will flow in parallel lines and are dependent of flow velocity, channel diameter, and fluid density.
- Laser Light: Monochromatic light or light of a single wavelength or frequency that is intense and small with the tendency to radiate as an almost non-divergent beam.
- Light Scatter: A light phenomenon that is composed of three independent processes that include diffraction, reflection, and refraction. The light scatter is actually in all directions. Diffraction is light that is bent at small angles relative to the light source. Refraction is that light bent at large angles to the light source.
- Linearity Limits: The instrument will be accurate as long as the results fall within a certain range known as the linearity range. Linearity ranges vary by instrument.
- Morphology: Description of cell size, shape, color, contour, etc., by light or electron microscopy or flow cytometry.
- Myelophthisic: Literal meaning consumption of marrow. It is the replacement of marrow by granuloma, tumor, or fibrous tissue (myelofibrosis), resulting in hypoproliferative marrow with presence of premature cells in peripheral blood.
- Myeloproliferative: Increased abnormal marrow production, it includes polycythemia vera, CML, essential thrombocythemia, etc., but not the acute leukemias.
- Optical Scatter: A cell counting principle that employs both laser and nonlaser light. The cell is identified and differentiated on the basis of light scatter and effects when it strikes the cell.
- Parameter: A statistical term that refers to any numerical value that describes an entire population.
- Random Access: Technological capability of an analyzer to process patient specimens independent of each other or to perform individual runs or panel of tests.
- Reagent Blank: The reagents and diluents (minus the patient specimen) that measure the degree of absorbance to eliminate false increases (due to reagent color) in the actual patient sample.
- Reflection: Forward light rays are bent or turned backward as the result of striking an obstruction.
- Refraction: The light beam or rays bend because of a change in speed and move in an oblique direction when passing from one medium into another because of changes in density.
- Scatter Plots: A two dimensional dot-plot histogram that allows definition of subpopulations of cells, in particular leukocytes.
- Sheath Fluid: Usually a 0.85% saline solution, it is the fluid that fills the flow cell and surrounds the sample stream as it passes through the flow cell. The sheath fluid keeps the flow cell clean, preventing the accumulation of reagents, cell debris, and any other substance onto the surface of the flow cell that would interfere with the laser light analysis. The sheath fluid also facilitates hydrodynamic focusing.
- Side Light Scatter: Also called “orthogonal light scatter”, this is laser light that is scattered away from the cell at a 90°degree angle. This is caused due to internal granularity of the cell. The more numerous the granules, the more there is side scatter.
- Threshold Limit: A limit imposed that will selectively eliminate or include cells of certain sizes to be counted. For the cell counter, it is the minimal electrical signal that will produce a pulse that will include a cell in the total count.