Hematology - Handbookfor Personal Use

Hematology Handbook Reference & Automation Methods
FIRST EDITION
Mohammed H. Saiem Aldahr, BSc (MT), MSc, PhD
Assistant Professor, Department of Laboratory Technology Vice Dean for Clinical Affairs. Vice Dean for postgraduate studies and scientific research. Faculty of Applied Medical Sciences, King Abdulaziz University Jeddah KSA
Hashem A. Abu-Hurirah, BSc (MT), MSc, PhD fellow

Application Manager Of Hematology Analyzer Application Manager Of Fluorescent Flow Cytometry Urine Analyzer PhD Fellow, Faculty of Science Faculty Of Allied Medical Technology Assistant Research King Abdulaziz University, Jeddah, KSA
Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE
Edited by: Bilal Khalaf, BSc Engineering, MBA 20 years of experience in Business Development, Operational Excellence (LEAN/6 Sigma), Marketing, Event Management and Training in Distribution, Healthcare, Education, Hardware and Software Industries.
To my late father, mother, wife and children, Heba, Osama, Ibrahim and Yousef for their support and encouragement. Hashem Jeddah 2011
Preface
Technology is on the rise and growing daily demands generate the need for instruments of this nature in the laboratory. They constitute a giant leap forward in the laboratory field, given the possibility of undertaking tests in a reliable, accurate, and precise manner, and delivering results in a shorter time period and under better control. The advantages of automation are numerous. Technology is continuously advancing to meet new developments in the field and to reduce turnaround times, allowing tests to be reliable, accurate, and precise, while maintaining quality. This work is done to give a user whole idea about the high end technology used in automated analyzer and reference methods to understand and be aware about the difference between automated principles and reference methodology. This handbook can be as a key to know the reference methods in laboratories, for medical applied students, laboratory technicians, technologist, medical technology students and laboratories trainees as well. This project is harvest of more than educational experience in hematology field, in than fifteen years of study and field experience student, technologist, sales, then marketing, twenty years of addition to more in hematology as application and
training in hematology field with different hematology companies merged to give useful and comprehensive information for the users of this hand book. The authors of this Hematology Handbook Reference & Automated Methods hope that the publication of this handbook will help technologists, technicians, and laboratories trainees in their assessment to be reference for blood counting procedures. This handbook has been compiled to help aid those involved to work in the field of medical laboratories, to skillfully assess the procedures and methodology which are used in hematology department and analyzers. INTENDED AUDIENCE This text is designed for medical applied students, laboratory technicians, technologist, medical technology students and laboratory trainees to get excellent idea about the high end technology used in automated analyzer and reference methods to understand and be aware of the differences between automated principles and reference methodology.
ACKNOWLEDGEMENTS I would like to acknowledge the work of Mr. Bilal Khalaf for his significant contribution in the design, editing and layout of the Hematology Handbook Reference & Automated Methods. I would also like to thank the entire Hematology Department team at King Abdulaziz University Hospital and Mr. Waleed Nasruddin for his scientific support in this project. Many thanks for my friends and colleagues Mohammed Moghrabi, Baha Bodyab, Mohammed Damati, Khaldoon Kasrawee & Jana Stark. Special thanks are due to Ms. Vicky Medina for her excellent secretarial help.
Hashem
Contents
Preface ......
1. 2. 3. Introduction ......... The Blood ........ Blood Collection.... IV 9 11 14
3.1. Capillary Blood..... 14 3.2. Venous Blood .... 15 3.3. Use Of Anticoagulants................................. 17 4. Blood Cell Counting ....... 19 4.1. 4.2. 4.3. 4.4. Red Blood Cell Counting........ White Blood Cells (WBCs) Counting.......... Platelets Count..... Reticulocytes Count................. 19 28 41 44
5. 6.
Automated Nucleated RBC Analysis............. Blood Film Examination.... 6.1. 6.2. 6.3. 6.4. 6.5. 6.6. Preparation Of A Thin Blood Film.... Preparation Of A Thick Blood Film....................... Staining Of Thin Blood Film With Wrights Stain............ Automated Digital Morphology... Test For Malaria..... Test For Leishmania..
48 49 49 50 50 53 54 55
7.
Hemoglobin Determination... 7.1. Cyanmethemoglobin Method. 7.2. Hemoglobinometer.
57 57 62 64 67
8. 9.
Hematocrit. Erythrocyte Sedimentation Rate (ESR)..
10. Sickle Cell Test.. 72 11. Coagulation... 74 11.1. Bleeding Time. 74 11.2. Prothrombin Time.. 77 11.3. Activated Partial Thromboplastin Time... 79 11.4. New Method Used In Semi-Automated & Full Automated Methods... 82 12. ABO And Rh Grouping... 13. Anti-Human Globulin (AHG) . 14. Atlas Of Hematology.... 89 103 115
Introduction
Hematology Handbook Reference & Automation Methods
Hematology is defined as the study of blood (blood forming tissues) and its components. Because of its critical role in maintaining life, blood has been referred to by some as the river of life. Additionally, because routine examination of the blood by means of the complete blood count (CBC) is the most widely performed test in the clinical laboratory and has also been referred to as a window on the body. Blood is one of the most complex organ systems in the human body. The key parts that make up the hematological system are the blood, bone marrow, spleen, and lymph system. In the adult, blood consists of approximately 55% plasma (liquid component) and 45% formed elements including: erythrocytes (red blood cells RBC), Leukocytes (white blood cells WBC), and (thrombocytes platelets). Blood makes up about 7% of the human body weight. Blood is formed from hematopoietic stem cells in the bone marrow. Blood, as a whole, is responsible for the most important functions
of life, such as the transport of metabolic components, nutrients, hormones, gas exchange, the immune defense, and coagulation. The science of hematology literally is part of the evaluation of all disease states. Blood, the fluid that nourishes and cleanses the body, has been at the center of interest and investigation from early humans to the field of modern science it has become today.
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2. Blood Cells and Platelets The blood is composed of three types of formed elements: erythrocytes (red blood cells), leukocytes (white blood cells) and platelets (thrombocytes). Erythrocytes: also called red blood cells (RBCs) are the body's most numerous blood cells. Average normal values of RBCs in the blood is about 4.60 x 106/L (4.60 million/ L) for female and 5.20 x 106/L (5.20 million/L) for male. RBCs play a major role in oxygen and carbon dioxide transportation through the circulation, as every cell contains approximately 280 million hemoglobin molecules and each molecule is attached with four iron atoms, which in turn combine with oxygen molecules by means of RBCs passing through the lung. Following this, RBCs travel through the circulation to the tissues where iron atoms release the attached oxygen into interstitial fluid and hemoglobin molecules take up carbon dioxide back to the lungs. This cycle keeps on repeating to maintain the body function and free the circulation from the harmful waste. Leukocytes: known as white blood cells (WBCs) and their function is defined in body defense mechanism against foreign agents and infections. Average normal range in the blood is between 4,500 to 11,000 cells per L. This value may be reported as 4.5-11.0 x 103/L or k/L (k = thousand). Unlike RBCs, WBCs are differentiated in different types. Most WBCs are filled with tiny grains and are called granulocytes (gran =
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grain). The normal range for granulocytes is 1.8-8.5 x 103/L of blood. These granulocytes are subdivided into the following: Neutrophils: 50%-70% of total WBCs or about 1.8-7.7 x 103/L Eosinophils: up to 5% of total WBCs or about 0.0-0.450 x 103/L Basophils: up to 2% of total WBCs or about 0.0-0.2 x 103/L
Another cell lineage is known as lymphocytes and monocytes which are classified as a granulocyte (nongranulocytic type of cells) as follows: Lymphocytes: 20%-47% of total WBCs or about 1.0 - 4.8 x 103/L Monocytes: 3%-10% of total WBCs or about 0.0 - 0.8 x 103/L
WBCs fight infection; some surround and destroy debris and foreign invaders, while others produce antigen/antibody reactions. An antigen is any substance (foreign or part of the body) which causes stimulation of antibodies production; subsequently, these antibodies neutralize antigens and such phenomenon is known as immunological humeral response. Important Considerations: The average adult circulation contains five liters of blood (roughly 5.28 quarts).
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Blood completes the entire systemic circuit from the left side of heart through the body to the right side of the heart in 90 seconds. Every cubic millimeter of blood contains five millions of RBCs. RBCs survive about 4 months and neutrophils survive about 6 hours in the blood stream.
Platelets: Known as (thrombocytes), they are cell fragments that travel in the bloodstream. The normal range for platelets count is 140 - 440 x 103/L. Platelets prevent blood and fluid loss by clumping together to begin the coagulation process. A blood clot is formed when sticky platelets become covered with fibrin (a plasma protein that holds the blood clot together). Each of the formed elements of blood will be covered in more details in the following sections. Hematopoiesis: The Formation of Blood Cells
All blood cells begin as undifferentiated stem cells capable of reproducing themselves. Generations of cells eventually differentiate into cell lines that will mature to produce erythrocytes, leukocytes, and platelets. Stem cells in bone marrow continuously proliferate usually at a steady state to maintain a constant population of mature blood cells. A disruption in this process can lead to serious illnesses.
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As a group, immature cells are large. As they age and mature, they become smaller and change in their reaction to the dyes used to stain them for identification. Role of the Spleen: Located beneath the diaphragm and behind the stomach, the spleen is an intricate filter that receives 5% of the total blood volume each minute. In the embryo, the spleen is a blood-forming center; it loses this function as the fetus matures. In the spleen, RBCs and WBCs are "inspected" by specialized WBCs called macrophages (disease fighting cells) which act to filter blood. Old or damaged cells are removed; salvageable cells may be "pitted", that is, unwanted particles are removed without destroying the cells. 3. Blood Collection 3.1 Capillary Blood Capillary blood is obtained from the tip of a finger in adults and from the great toe or the heel in infants. The area where the blood is to be collected must be cleaned with 70% alcohol, dry with sterile gauze, and puncture the skin with a sterile disposable blood lancet that is designed to penetrate no deeper than 2mm. Use a sterile gauze to wipe out the area for blood collection since it will alter the composition of blood specimens. If blood collection is difficult, warm or allow it to remain in hanging position for some time.
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Advantages: a. Capillary blood can be obtained with ease. b. Capillary blood is the preferred material for making peripheral blood smear. Disadvantages: a. Only a small specimen can be obtained and repeated examinations require new specimens. b. Blood in micro-tubes frequently haemolyses, resulting in interferences with most laboratory tests. c. Test results on capillary blood cannot be compared with test results in venous blood. d. The finger is not only sensitive but difficult to adequately sterilize in the time usually available. In patients with lowered resistance to infection, a specimen taken from the finger by capillary is much likely to lead to infection than one taken from the veins. e. Red and white cell counts and enumeration of platelets and Reticulocytes should not be performed on capillary blood because of the difficulty in standardizing capillary blood flow. 3.2 Venous Blood Venous blood is mandatory for most tests that require anticoagulation or larger volume of blood, plasma or serum that can be provided by capillary blood. Although other veins can be chosen, venous blood is usually obtained
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from one of the capital fossa veins. The vein is congested by placing a tourniquet on the upper arm and tightens it sufficiently to retain venous blood and prevent it from return. Following this, the arm must be disinfected thoroughly using (70% Ethyl Alcohol), once the blood is started to rush out from the vein into the collecting tube, loosen the tourniquet and obtain blood by gentle suction. At the end, use sterile gauze to apply over the puncture site. Remove the needle from the syringe, and quickly transfer blood into a test tube which may or may not contain anticoagulant. If an anticoagulant has been added, mix blood gently by inverting the stoppered tube several times. Advantages: a. Multiple and repeated examinations can be performed on the same specimen. b. Aliquots of the specimen may be frozen for future reference. c. There is no variation in blood values, if specimens are collected from different veins. Disadvantages: a. The venous method is somewhat lengthy procedure that requires more preparation than the capillary method. b. Prolonged stasis produced by the tourniquet must be avoided, because it results in haemoconcentration.
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3.3 Use of Anticoagulants Anticoagulant: Anticoagulant is a substance that prevents coagulation; (stops blood from clotting). A group of pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic disorders. Some chemical compounds are used in vitro such as; Ethylenediamine Tetra-Acetic (EDTA), Trisodium Citrate, Ammonium Oxalate, Potassium Oxalate and Heparin. a. Ethylenediamine Tetra-Acetic Acid (EDTA) It is a sodium or potassium salt of Ethylenediamine Tetra-Acetic Acid which prevents the blood from clotting by removing and binding calcium. EDTA can be used for both hematology and some chemistry tests. It is considered an excellent anticoagulant and it is widely used. The amount of EDTA required for 10cc or less of blood is 10mg of the dry powder or 0.1cc of a 10% solution. The EDTA in the test tube is not necessarily to be evaporated; it may be used in liquid form. The following Table discussed the time of blood collected in EDTA.
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Table 3.1: Approximate keeping time for blood anticoagulated with EDTA Test White Blood Cell Count Red Blood Cell Count Hemoglobin Determination Stained Red Cell Examination Sedimentation Rate Hematocrit Reading Reticulocyte Count Red Blood Cell Indices Platelets Count Blood Grouping Differential White Cell Count b. Trisodium Citrate Trisodium citrate prevents the blood from clotting by removing and binding calcium. The ratio of blood to sodium citrate solution is 9:1 i.e. for 9ml blood add 1ml sodium citrate solution for coagulation tests 4:1 for ESR estimation. c. Mixture of Ammonium Oxalate and Potassium Oxalate This mixture of oxalate salts prevents the blood from clotting by removing and precipitating calcium. This
Keeping Time 24 hr 24 hr 1 hr 1 hr 2 hr 24 hr 1 hr 3 hr 1 hr 48 hr 1hr
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anticoagulant should not be used in chemistry tests such as Urea and Potassium. d. Heparin Heparin prevents the blood from clotting by neutralizing thrombin. It is used at a concentration of 10-20 IU/ ml of blood. Heparin is the best anticoagulant to use for osmotic fragility tests. However, heparinized blood should not be used for making blood films. 4. Blood Cell Counting: RBC count and indices (MCV, MCH, MCHC) 4.1 : (RBC) Count Reference and Automated Methods Reference Method Measurement of the total number of circulatory RBCs is important because the number of RBCs and the amount of hemoglobin, they contain, must remain within certain limits to maintain good health. The main function of the red cell is to carry oxygen from the lungs to the body tissue and to transfer carbon dioxide from the tissue to the lungs. The process is achieved by the (hemoglobin) found in the red cells that combine easily with oxygen and carbon dioxide.
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Equipment: a. 10ul automatic pipette or Red cell pipette (Figure 4.1). The red cell diluting pipette contains a red bead in the bulb and has ten divisions on the capillary end, with points 0.5 and 1.0 numbered. If the blood is drawn to the 0.5 mark and the pipette filled with diluting fluid, the resultant dilution is 0.5:100 or 1:200, the dilution used in routine counting. One volume of diluting fluid remains in the stem does not enter the bulb, is blown out first the counting chamber filled and therefore does not dilute the blood. b. Counting Chamber (Haemocytometer). The most commonly used method employs the Improved Neubauer counting chamber (Figure 4.2). There are two Chambers per Haemocytometer, each consists of nine large squares and measuring about 1mm2 as demonstrated in (Figure 4.2).
Reagent: Diluting Fluid Trisodium citrate Concentrated formaldehyde (37%) Distilled water
3g 1ml 100ml
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Procedure: 1. Add 10ul of the patient whole blood sample to 2ml of red cell solution. If red cell pipette is used, draw blood to 0.5 marks and complete with red cell solution to 101 marks. 2. Mix well the diluted blood (The dilution of blood is 1:200). 3. Discard the first 5 drops before charging the counting chamber, if red cell pipette is used. 4. Fill the counting chamber and be careful not to overfill beyond the ruled area, and check if air bubbles present. 5. Place the chamber on the stage of the microscope and use 40X objectives. 6. Count the cells in five squares of red cell count area (R) as shown in the figure 4.3.
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Figure 4.1: Explanation of dilution of blood in red and white blood cells counting pipettes.
Figure 4.2: Improved Neubauer haemocytometer counting chamber ruling.

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Figure 4.3: Demonstrates the Counting chamber rulings and dimensions. Haemocytometer of counting chamber has two ruled areas etched on its surface, each consisting of a 3mm square divided into nine large squares (W) and each measure 1mm 2. Center square, which is used for red cell counting, is subdivided into 25 smaller squares (R), each occupying an area of 0.04 mm 2. Red cells in the five squares are enumerated. The depth of the chamber is 0.1mm and the four large corner squares are subdivided into 16 smaller squares and are used for leucocyte counting. Each measures 1mm2 in area.
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Figure 4.4: Improved Neubauer ruling for one counting chamber. White cell count is done on the four large corner squares (1, 2, 3 and 4) of each of two counting chambers.
Calculation: Each R section (Fig. 4.2) has an area of 0.04mm2 and depth of 0.1mm. The volume of 1 R is found as follows: Area of 1 R x Depth of 1 R = Vol. of 1 R 0.04mm2 x 0.1mm = 0.004mm3
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Number of R sections x Vol. of 1 R = total Vol. 5 x 00.004 = 0.02mm3 Vol. Correction Factor = Vol. Correction Factor = Vol. Desired Vol. Used = 50
1.0mm3 0.02mm3
Red Cell Count = No. of cells in 5 R x dil. factor x Vol. Correction (Per cu.mm) = Number of cells x 200 x 50 = Number of cells x 10000
Expected Range Males Females Children 4.5-5.5 x 106/cu.mm 4.0-5.0 x 106/cu.mm 4.2-5.2 x 106/cu.mm
Red Cell Indices:
i. Mean Cell Volume (MCV) The mean cell volume is the volume of average erythrocyte.
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Normal range: 76-96 fl MCV = PCV x 10 RBC Count in Million
ii. Mean Cell Hemoglobin (MCH) This is a calculation of the ratio of hemoglobin to the erythrocyte count. The formula for the calculation is: MCH = Hb (g/dL)/RBC (x 106/L) x 10 iii. Mean Cell Hemoglobin Concentration (MCHC) This formula is the calculation of the ratio of hemoglobin to hematocrit that indicates the concentration of hemoglobin in the average red cell. The calculation is: MCHC = [Hb (g/dL)/Hct (%)] x 100 Automated Method Direct Current (DC) Technology: Also it is known as Impedance counting or Coulter Principle (Electrical Sensing Zone Method) has become the accepted "Reference Method" throughout the world for particle size analysis and is the recommended limit test for particulate matter in large-volume parenteral solutions.
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This method of sizing and counting particles is based on measurable changes in electrical resistance produced by nonconductive particles suspended in an electrolyte. A small opening (aperture) between electrodes is the sensing zone through which suspended particles pass. In the sensing zone each particle displaces its own volume of electrolyte (Figure 4.5). Volume displaced is measured as a voltage pulse; the height of each pulse being proportional to the volume of the particle. The quantity of suspension drawn through the aperture is precisely controlled to allow the system to count and size particles for an exact reproducible volume. Several thousand particles per second are individually counted and sized with great accuracy. This method is independent of particle shape, color and density.
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Figure 4.5: Explanation of Direct current small opening (aperture).
4.2 White Blood Cell (WBC) Count Reference Method Measurement of the total number of circulating white blood cells (WBCs) is an important procedure in the diagnosis and prognosis of the disease process. The main function of leucocytes is to fight infection by phagocytosis and production of antibodies. Since leucocytes are affected by so many diseases, the leucocyte count serves as a useful guide to the severity of the disease process. The increase of white blood cells above
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10x109/L is called leucocytosis and the decrease of white blood cells below 4x109/L is called leucopenia. Reagents Diluting Fluid: Acetic Acid (Glacial) 4ml Distilled Water 200ml Aqueous methylene blue (0.3gm %) 20 drops Procedure 1. Add 50ul of whole blood sample to 0.95ml of diluting fluid into a labeled test tube. If you use white cell pipette is being used (Fig. 4.3), draw blood to 0.5 marks and continue with the diluting fluid to 11 marks. 2. Shake well (The dilution of the blood is 1 in 20). 3. Prepare the counting chamber and attach the cover glass by pressing it carefully into place. 4. Discard first 4 drops if you are using white cell pipette. 5. Fill the counting chamber (take care not to overfill beyond the ruled area). 6. Allow the cells to settle for 3 minutes.
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7. Place the chamber on the stage of the microscope, use the 10X objective, and reduce the light by lowering the condenser. 8. Count WBC in 4 big (W) squares at the corners of the counting chamber (see Fig IV.2). Making the Calculations Each W section (Fig. IV.2) has an area of and depth of 0.1mm. The volume of W = 1mm2 x 0.1 = 0.1 cu.mm The volume = number of W x volume of W Vol. correction = 1 cu.mm = 2.5 0.4 cu.mm
Number of WBCs = Number of cells in 4 W (0.4 cu.mm) x dilution Fac. x correction factor Number of WBCs = Number of cells in 4 W (0.4 cu.mm) x 20 x 2.5 Expected Range 3 - 10 x 103/cu.mm
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Common Sources of Error a. b. c. d. Failure to have required blood volume. Failure to mix well. Failure to discard the first 4 drops. Failure to properly charge the counting chamber.
The least Frequent Sources of Error are a. b. c. d. e. f. g. h. i. j. Inaccurate pipette or counting chamber. Moist or unclean pipette. Excessive pressure in finger when obtaining the blood. Excessive or insufficient dilution of the fluid. Slowness in manipulation, thus, allowing the blood to clot. Air bubbles in the pipettes. Air bubbles in the counting chamber. Presence of yeast or other contaminants in the diluting fluid. Presence of many nucleated red cells causing a high white cell count. Miss counting or calculations.
Correction for Nucleated Red Cells In some anemia, such as thalassaemia and erythroblastosis fetalis, many nucleated red cells may be found in the blood. Since these nucleated red cells are not dissolved by the white cell diluting fluid, they are counted as white cells.
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This would result in an erroneously high white cell count. Consequently, the count must be corrected. If large numbers of nucleated cells are found in the stained red cell examination, correction of the white cell counts must be done as shown below: Corrected WBC = Uncorrected WBC x Where: 100 = White cells counted in the differential white cell count. A= Number of nucleated red cells counted while counting the 100 white cells of the differential count. 100 100 + A
Automated Method There are more than one way to count WBCs with different Techniques, these techniques are established and applied by different companies. a. Using Radio Frequency (RF) and Direct Current (DC) to enumerate and identify WBC populations. The RF method detects and sizes lyse-treated cells based on density and nuclear size, whereas the DC method sizes the entire cell, nucleus, and cytoplasm. Cells pulses detected by RF and DC methods are then displayed as a 3dimensional WBC scatter gram which contains
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b.
information about the distribution of lymphocytes, monocytes and granulocytes. Separate histograms are generated for basophil and eosinophil populations. The best way to get the maximum accuracy is counting of WBCs by different channels using of multichannel method especially in abnormal samples.
Counting of total WBCs differential by using: 1. Size /volume of cells DC detection method Laser light scatter 2. Internal structure (density or complexity), e.g. granulation Flow cytometry with laser light scatter 3. Nucleic acid content (RNA and DNA) Fluorescence Flow Cytometry with laser light scatter
By using this method, the Size of blood cells is not relevant in the Differential counting channel. Debris do not interfere, also cells without nucleic acids such as mature Erythrocytes are not stained, no interference by lyse-resistant RBC, and non-cellular particles such as air bubbles are not detected. (Figure 4.7)
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Figure 4.6: Explanation of optical system (Laser light scatter)
Figure 4.7: Explanation of side fluorescence, side scattered and forward scattered light in laser flow cytometry
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c. Volume Conductivity and Light Scatter (VCS), performs a five-part differential consisting of monocyte, lymphocyte, granulocyte, eosinophil and basophil. Established WBC differential technology uses three measurements: individual cell volume, high-frequency conductivity and laser-light scatter as explained below: Volume Analysis - Electronic leukocyte volume analysis uses a low-frequency current measure volume of the WBCs as they impede the current when they pass through the aperture. Conductivity - Cell walls act as conductors to highfrequency current. As the current passes through the walls and through each cell interior, it detects differences in the insulating properties of cell components. It characterizes the nuclear and granular constituents and the chemical composition of the cell interior. Scatter - Leukocytes are hydrodynamically focused and passed in a steady stream through a sensing zone on which a laser light is focused. As each cell passes through the sensing zone of the flow cell, it scatters and reflects the focused light which is detected by a photodetector. The patterns of scatter are measure at various angles (forward scatter at 180 degrees. and right angle scatter at 90 degrees). Scattered light provides information about cell structure, shape and reflectivity. The characteristics are used to differentiate the various
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types of WBCs and to produce scatter plots with a fivepart differential. d. By using of optical flow cytometry, cytochemistry and light scattering and staining with peroxidase technologies to enumerate and identify cells. WBCs are fixed with formaldehyde and stained with peroxidase in the peroxidase reaction chamber. The high heat in the chamber lyses platelets and RBCs and causes the WBCs to be fixed and dehydrated. Forward-angle light scatter and tungsten light optics are used to measure WBC size and peroxidase activity. Myeloperoxidase is a granulocyte enzyme marker that is present in varying degrees in neutrophils, eosinophils and monocytes but is absent from basophils, lymphocytes and blasts. A specific basophil count is determined separately in the basophil/lobularity chamber. Whole blood is exposed to an acid buffer that selectively lyses all cells except basophils. The resulting particles are sorted and quantified by measuring the forward angle and light scattering properties. An additional category of cells is reported as LUC (large unstained cells). This category reflects atypical lymphocytes or blasts. e. System of Multiangle Polarized Scatter Separation (MAPSS) flow cytometry with hydrodynamic focusing of the cell stream. The leukocyte differential is accomplished by light scatter. It features three independent measurements and focused flow impedance.
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Multidimensional light scatter and fluorescent detection are used as well. DIFFERENTIAL WBC AUTOMATED COUNTERS 3-Parts differential counting machines Small automated machine with 8, 12, 16 or 18 parameters screening machine for small, satellite laboratories, emergency rooms, etc. some manufacturers count monocyte, lymphocyte and mixed (granulocyte; neutrophile, basophile and esinophile), but the others count lymphocyte, neutrophile, and mixed (monocyte, basophile and esinophile) to get an idea for the patient's status (viral or bacterial infection). 5 Parts differential counting machines Medium and large fully automated machine with up to 80 parameters, consider as a diagnostic machine for hospitals. Immature granulocyte (IG):
Immature granulocyte (IG) which includes: myelocyte, promyelocyte and metamyelocyte, recently has been considered as the 6th part differential in differential counting.
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Figure 4.8: Explanation of the 5 part differential WBCs find in differential BC count and band cells
Neutrophilic Segmented Cell Neutrophilic Band Cell
Size: medium Nucleus: broken up into segments Cytoplasm: contains small pink or brownish granules Comments: May be confused with a neutrophilic band cell. When in doubt, call cell a neutrophilic segmented cell. When found: 55 to 75% in normal blood; increased in appendicitis, pneumonia.
Size: medium Nucleus: shaped like a band Cytoplasm: contains small pink or brownish granules Comments: May be confused with neutrophilic segmented cell. When in doubt, call cell a neutrophilc segmented cell. When found: 2 to 6% in normal blood; increased in appendicitis, pneumonia.
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Eosinophilic Segmented Cell
Basophilic Segmented Cell
Size: medium Nucleus: usually has 2 lobes or segments Cytoplasm: contains large red granules Comments: Eosinophilic segmented cell has large red granules whereas neutorphilic segmented cell as small pink or brownish granules When found: 1 to 3% I normal blood: increased in asthma, hay fever, etc.
Size: small or medium Nucleus: usually indistinct; appears buried under large purple or purplishblack granules Cytoplasm: contains large purple or purplish-black granules Comments: Easily identified by the large purple or purplish-back granules scattered throug hout the cell. When found: 0 to 1% in normal blood.
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Lymphocyte
Monocyte
Size: small, medium, or large Nucleus: closely knit and usually round Cytoplasm: light blue; may contain a few reddish granules, cytoplasm may be sparse and even absent in some small lymphocytes Comments: Large lymphocyte may be confused with monocyte. Nucleus of large lymphocyte is closely knit and usually round. Nucleus of monocyte is spongy and sprawling. When found: 20 to 35% in normal blood; increased in infectious monocleusis, lymphocytic leukemia, and many other diseases.
Size: large Nucleus: spongy and sprawling Cytoplasm: light grey; may contain very tiny reddish granules Comments: Monocyte may be confused with large lymphocyte. Nucleus of monocyte is sponsry and sprawling. Nucleus of lymphocyte is closely knit and usually round When found: 2 to 6% in normal blood; increase in tuberculosis and monocyte leukemia.
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4.3 Platelet Counts Reference Method Platelets are the smallest elements in the blood. These cells are non-nucleated round or oval shaped cells. Platelets activity is necessary for blood clotting therefore, deficiency in these cells will lead to prolonged bleeding time. The life span of a platelet is approximately 5-7 days. Abnormally increased number of platelets occurs in cancer, splenectomy, iron deficiency anemia, and cirrhosis, while abnormally decreased number of platelets occurs in idiopathic thrombocytopenic purpura (ITP), pneumonia, allergic conditions, infections and toxic effects of certain drugs. Reagents Diluting fluid (1% Ammonium Oxalate) Ammonium oxalate 1g Distilled water 100ml Procedure a. b. c. d. Pipette 0.95ml of diluting fluid into a labeled test tube. Add 50ul (0.05ml) of the patient whole blood sample. Shake well. The dilution of the blood is (1) in (20). Fill one side of a Neubauer counting chamber and allow the platelets to settle for 10-20 minutes in a moist petridish. e. Count the same area as for red cell count.
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The platelets will appear as small refractile bodies under the x 40 objective under microscope. Calculation Platelet Count = No. of cells in 5 R x dil. Fac x fac Vol. Corr (per cu.mm) (Figure IV.2): = No. of cells x 20 x 50 Expected Range 150 - 400 x 103/cu.mm Automated Method The four main procedures for platelet counting are: manual phase contrast microscopy, impedance, optical light scatter/fluorescence and flow cytometry. Early methods to enumerate platelets were inaccurate and irreproducible. The manual count is still recognized as the gold standard or reference method, and until very recently the calibration of platelet counts by the manufacturers of automated cell counters and quality control material was performed by this method, impedance counts still have limitations as cell size analysis cannot discriminate platelets from other similar-sized particles. More recently, light scatter or fluorescence methods have been introduced for automated platelets counting (Fluorescenceoptical: PLT-o = RNA content), fluorescent stain is used to detect the residual amount of RNA (Figure 4.10), but there are
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still occasional cases where an accurate platelets count remains a challenge. Thus, there has been interest in the development of an improved reference procedure to enable optimization of automated platelet counting (Figure 4.10). Monoclonal antibodies to platelet cell surface antigens conjugated to a suitable fluorophore is costly method, permits the possible implementation of a new reference method to calibrate cell counters, assign values to calibrators, and to obtain a direct platelets count on a variety of pathological samples. In future, analyzers may introduce additional platelet parameters; a reliable method to quantify immature or reticulated platelets would be useful.
Figure 4.9: Explanations one of the procedures used in PLT counting by using Tungsten Lamp to detect light absorbance and light scatter
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Figure 4.10: Explanations the detection the residual amount of RNA in platelets
4.4 Reticulocytes Count
Reticulocytes are juvenile red cells that contain fine, deep violet granules (remnants of the ribosomes and the ribonucleic acid present in the precursor cell) arranged in a network. Principle Reticulocytes are immature red cells that pass into the blood stream from the bone marrow. The number of reticulocytes in the blood indicates the degree of activity of the bone marrow. The number increases when the marrow is very active.
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Reference Method Reagent (Saturated Solution of Brilliant Cresyl Blue) Brilliant cresyl blue Trisodium citrate Sodium chloride solution (0.85%) Procedure a. Filter a small amount of the cresyl blue solution into a test tube. b. Add equal quantity of venous blood collected in EDTA di-potassium salt. c. Mix gently and leave for 10 minutes at 37C or 15 minutes at room temperature. d. Shake the tube and make a thin smear of the mixture. e. Examine the smear using the oil-immersion objective in microscope. f. Examine at least 1000 red cells. g. Count the total number of red cells and the number of reticulocytes. h. Calculate the percent of reticulocytes as follows: Number of reticulocytes 1000 X 100 1.0g 0.4g 100ml
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Note: In order to decrease the microscopic field and thus make it easier to count the cells, place a piece of paper containing a Window in the eyepiece of the microscope as shown in this (Figure 4.11).
Figure 4.11
Reference values Adults & children Infants 0.2 - 2.0% 2 - 6%
Conditions accompanied with abnormal reticulocytes count as seen in the following Table 4.1. Reticulocytosis Hereditary spherocytic anaemia Sickle cell anemia Thalassaemia Paroxysmal noctoral Hb-Uria Acquired autoimmune Hem-anemia Acute posthemorrhagic anemia Reticulocytopenia A plastic anemia Pernicious anemia
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Automated Reticulocyte Analysis Can be measured by three different methods a. Fluorescence based: Size and RNA/DNA content of cells: (Reticulocyte channel) The residual amount of RNA is measured by using fluorescent stain to prevent RBC derbies, crystals or protein interferences. b. The exceptional capability of Volume Conductivity and Light Scatter (VCS) Technology in which simultaneous measurements of cell-by-cell characteristics are determined using the three distinct energy probes of volume, conductivity and laser light scatter is used to interrogate red cells with the same thoroughness that has made VCS technology the right choice for more laboratories than any other. Red cells are evaluated not only in terms of size, using the DC technology, but also by the degree of opacity and log light scatter. The Log Light Scatter differentiates the reticulocytes from mature red cells. Immature reticulocytes with more RNA scatter more light and appear further to the right in the reticulocytes population in the scattergram.
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c. Nucleic acid dye Oxazine 750 is used to stain isovolumetrically sphered cells then the analysis by the laser optical assembly. Additional Reticulocytes parameters All Parameters determined for RBCs are separately analysed also for Reticulocytes (MCVr, CHCMr, RDWr, HDWr, (CHr), CHDWr), immature reticulocytes Fraction, (IRF), low fraction reticulocytes (LFF), medium fraction reticulocytes (MFR) high fraction reticulocytes (HFR) and mean reticulocytes volume (MRV). Reticulocyte haemoglobin (RET-He) or haemoglobin content of reticulocytes (CHr) has been shown to be a sensitive direct measurement (monitor EPO and iron therapies). 5. Automated Nucleated RBC analysis Measurements were performed in the extended lyse mode because erythrocytes in newborns are relatively resistant to the lysis agents, uses a semiconductor laser. The cell membranes of NRBC and mature erythrocytes are lysed, while the WBC become permeable, allowing quick influx of the dye, but remain intact. The nuclear material of NRBC and WBC is stained with a polymethine-based. Thus two clear populations emerge on the basis of differences in fluorescence intensity. Using forward scatter light signals, volumetric
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differences between nucleated cells and ghosts permit their clear distinction. Uses an argon laser in combination with two fluorescence channels to measure NRBC. The reagent lyses the membrane of NRBC and mature erythrocytes, while WBC are left intact. The fluorochrome propidium iodide binds to the nuclear DNA of the NRBC and WBC. The combination of cell size and fluorescence intensity permits the separation of NRBC from the leucocyte population. The absolute NRBC count and theproportion of NRBC per 100 WBC are reported.
6. Blood Film Examination 6.1 Preparation of a Thin Blood Film a. Collect a drop about 3-4 mm in diameter at one end of the slide. b. Hold the slide with one hand and place the edge of the spreader just in front of the drop of the blood using the other hand. c. Draw the spreader back until it touches the drop of blood. d. Let the blood run along the edge of the spreader. e. Push the spreader to the end of the slide with a smooth movement (all the blood should be used up before reaching the end).
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f. Check if the blood film is satisfactory by the following: There should be no lines extending across or down through the film. The film must be smooth at the end, not ragged and lined. The film must not be too long. The film must not be too thick. The film must not contain holes because a greasy slide has been used.
6.2 Preparation of a Thick Blood Film a. Collect 3 drops of blood about 3-4 mm in diameter at the centre of the slide. b. Quickly join the three drops of blood using the corner of the spreader, and spread them to make an even, thick film. The blood should not be excessively stirred but can be spread in a circular or rectangular form with 3-6 movements. 6.3 Staining of Thin blood Film with Wrights stain The stained thin blood films can be used to study the morphology of RBCs, WBCs and platelets, besides the WBCs differential count.
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Principle Wrights stain is a methyl alcohol solution of an acid dye and a basic dye. The acid is known as eosin, it is red in color. The basic is known as methylene blue, it is blue in color. In the staining process, a buffer solution is used to control the acid-base balance of the stain. Reagents a. Wrights stain solution b. Phosphate Buffer (pH 6.8) Eosine 0.3% (W/V) Methylene blue Na2HPO4 KH2PO4
Procedure 1. Prepare a thin blood film. 2. Fix the film by absolute methyl alcohol for 2-3 minutes. The smear will change from red to light brown. 3. Completely cover the slide with stain. After 3 minutes, add an equal volume of buffer. Blow by using hair dryer gently to ensure uniform mixing. A green metallic sheen will appear. 4. After an additional 5 minutes rinse thoroughly with tap water. Wipe the back of the slide to remove all traces of stain. Air dry the slide and add a drop of immersion oil.
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5. Use the X40 & X100 objectives in the microscope to examine the blood film for cells por-mpohology and for WBCs differential (Figure 6.1).
Figure 6.1: Method of examining the blood smear for the differential white cell count. When a blood smear is made, the large cells tend to accumulate on the edge of the smear, whereas the small cells on the middle of the smear, it would not be a true representative sample of the patients cells. Therefore, the smear is examined by following the path of the arrow shown above (Fig 6.1).
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Figure 6.2: Explanation of the monolayer area of the blood film where WBCs and platelet numbers are estimated and cell morphology is examined.
6.4 : Digital Morphology After slide staining digital morphology system will read the slide and reported back for approval, automatic cell location and pre-classification improves resource utilization, the quality of results and employee satisfaction. One particular labor benefit is that highly skilled medical technologists are able to spend more time on difficult cases that require careful analysis and assessment. The ability to archive images enables hospitals to look at previous cell images of patients in case of relapse. An additional service is the ability to create digital slides.
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Although primarily designed for hematology smears, the digitization function is also applicable to other types of samples, for educational purposes. Advantages of automated image analysis as compared to manual analysis Increased productivity (speed and cost-efficiency). Quality assurance (competence assurance and standardized results). Ability to track information. Improved conditions for sample assessment by enhanced communication with other information systems and instruments in the laboratory, giving the user more access to information. Improved cooperation within and between laboratories (transfer of digital images to experts outside the laboratories). Improved ergonomics (eyes, neck, and back).
6.5 Test for Malaria The most commonly used technique for blood examination of malaria is stained blood films. Giemsa stain (3% Giemsa solution in buffered distilled water, pH 7.2) is usually used to stain the films. For routine malaria microscopy, a thin and a thick film are made on the same slide.
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In examining thin blood films for malaria, infected red blood cells and the parasites inside the cells might be identified. In stained thick blood films, the red blood cells are lysed. Thus, diagnosis is based on the appearance of the parasite. In thick films, organisms tend to be more compact and denser than in thin films (Figure 6.3 A, B and C).
6.6 Test for Leishmania The parasites are most easily obtained in slit-skin smears from the nodular edge of the sore, which is held between finger and thumb to cause blanching. Using a small scalpel blade, an incision a few millimeters along is made through the intact epidermis into the dermis and the exposed surface is scraped to obtain tissue juice and cells. Smears are stained with Giemsa or another equivalent stain and examined directly. Smears that contain blood, pus, or epithelial debris are unusable. Smears are stained with Giemsa stain and examined under oil-immersion (see illustration below). The pH of the buffer saline used in the Giemsa stain should be 6.8 for Leishmania (Figure 6.3 D).
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Figure 6.3
A. Malaria
B. Malaria Vivax
C. Malaria plasmodium falciparum
D. Leishmania promastigotes
The parasite density is graded according to the following (Table 6.1). Grade 6+ 5+ 4+ Average parasite density >100 parasites/field 10-100 parasites/field 1-10 parasites/10 field
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3= 2+ 1+ 0
1-10 parasites/10 field 1-10 parasites/100 fields 1-10 parasites/1000 fields 0 parasites/1000 fields
7. Hemoglobin Determination 7.1 Cyanmethemoglobin Method Hemoglobin is the main component of red cells. It is composed of two pairs of globin chains and a hem compound which contains iron. The main function of hemoglobin is to carry oxygen (O2) from the lungs to the body tissue cells and to transfer carbon dioxide (CO2) from the tissue cells to the lungs. The oxygen combining capacity of the blood is directly proportional to the hemoglobin concentration rather than to the RBCs count. The hemoglobin determination is useful to screen and measure for the severity of anemia and to follow its response to treatment. Principle The basis of the method is dilution of blood in a solution containing potassium cyanide and potassium ferricyanide. Hemoglobin, Methemoglobin and Carboxyhemoglobin are all converted to Cyanmethemoglobin. The absorbance of the
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solution is then measured in a photoelectric colorimeter or spectrophotometer at a wavelength of 540nm. Sample Blood may be taken directly from a finger (or heel) puncture without use of anticoagulant or may be collected in a test tube (EDTA, K3) as an anticoagulant. Reagents a. Drabkins Reagent Potassium Ferricyanide K3 Fe (CN)6 = 0.2g Potassium Cyanide KCN = 0.05g Sodium Bicarbonate NaHCO3 = 1.0g
Procedure One reagent blank per series is required. Table 7.1: Illustrates the Cyanmethemoglobin Method for Hemoglobin Determination Reagent Drabkins solution Hemoglobin standard Sample (blood) Blank 2.5 ml Standard 2.5 ml 10 ul Test (sample) 2.5 ml 10 ul
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Mix thoroughly all blood specimens to be tested by repeated inversion immediately before testing. Mix and let stand for approximately 5 minutes to ensure complete reaction. Read using spectrophotometer at a wavelength of 540nm against blank. Calculation Cb g / dl = Ab As x Cs
Cb = The concentration of Hemoglobin in a given sample Ab = The absorbance of the sample As = The absorbance of the standard Cs = The concentration of the standard Hemoglobin g/dl x 0.62 = Hemoglobin (Fe) mmol/L.
Example First try to adjust the spectrophotometer machine to read (0.00) absorbance against a blank in all of the following examples. a. Absorption Mode For determination of Hemoglobin in an unknown sample, the following results were obtained.
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Concentration (Cs) of Hemoglobin standard = 15g/dl Reading (As) of Hemoglobin standard = 0.45 Reading (Ab) of unknown (sample or control) = 0.34 Cb (g / dl) = Concentration of unknown (g/dL) Ab As = x Cs 0.34 0.45 x 15 = 11.3
b. Concentration Mode o Press mode selector in the spectrophotometer until the concentration lid is lit. Place the standard solution in sample compartment. o Using the Concentration/Factor Adjust Control, set the concentration value of the standard (15g/dl) on the digital display. o Insert the samples in the sample compartment and read results in the concentration unit. c. Factor Mode If the factor is already known for the test, usually enter this value by: o Pressing the mode until the factor lid is lit. o Using the Concentration/Factor Adjust Control to set the display to the desired factor value (33).
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o Insert the samples in the sample compartment and read results directly in concentration unit. Calculation of the Factor Cb = Factor (F) = Ab As Cs As x Cs = 15 0.45 =3
Cb = F x Ab = 33 x 0.34 = 1.3mg/dl Reference values Adult Male Adult Female Newborn 13.5 18g/dl 12 16g/dl 16 20g/dl
Cyanide is toxic chemical compound, the new automated methods using the same principle but in different reagents instead of Drabkins Reagent (e.g sodium laurryl sulfate or organic quaternary ammonium salt with sodium chloride). Standard Cyanmethermoglobin standard: The concentration is usually given as g/ dl.
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Note: Cyanide is a well known as a lethal chemical; therefore, reasonable care must be exercised in handling this solution. 7.2 : Hemoglobinometer (CYNOX I) Cynox I Hemoglobinometer is a photometer designed to provide accurate hemoglobin determinations using a 1.251 dilution with a readout in g/dl or mmol/L. Method a. Place the on-off power switch in either the up or the down position to activate the instrument. Allow one minute for warm up. b. Adjust the zero control after inserting reference cuvette into the hemoglobinometer to obtain a display reading of 0.0. c. Open the cover and insert a calibration reference slide into the slide compartment matching the white dots. (Glass slides should be free of dirt, oil or fingerprints). d. Close the cover and compare display reading against value provided with the calibration of reference standard. (Standard reading should agree within 0.5 units). e. Dispense 5ml of Cynox 1 reagent or Drabkin reagent into a clean dry test tube.
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f. Draw blood until the 0.02ml end to end capillary is completely filled. Wipe off excess blood on the outside very carefully. g. Drop the capillary into the reagent, cover the end of the test tube and shake horizontally until no blood is left in capillary. h. Transfer the resultant solution to a clean dry cuvette bringing the level to approximately full. Clean the outside of the cuvette. i. Place the cuvette containing diluted blood sample into the cuvette compartment. j. Close the cover and obtain the hemoglobin value from the digital display. Automated Method In cell blood counters the principle is same but potassium cyanide almost is not still used. Separate Hemoglobin channel ensure reliable results to prevent interference of Leukocytes is minimized by means of a cyanide-free reagent (Figure 7.1).
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Figure 7.1: Hemoglobin measurement channel in Automated Method. 8. Haematocrit (Packed Cell Volume, PCV) This is one of the simplest, most accurate and most valuable of all hematological investigations. By means of haematocrit, hemoglobin and red cell count the absolute indices can be calculated. High speed micro haematocrit centrifuge has become commercially available; it provides a centrifugal force of about 12,000 g which gives a constant packed cell volume after a 3minute centrifugation.
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Reference Method a. Capillary or venous blood is drawn into 75mm long; 1.5mm bore heparinized capillary tubes from finger, lobe of the ear or the heel (infants). b. Seal the capillary tube at the end with a commercially available sealing compound or by heating the end of the tube over a spirit lamp. c. Place the capillary tubes in the numbered slots in the centrifuge head, making sure that the number of the slot corresponds with the specimen number. The end of the sealed tube should point outward away from the centre. d. Centrifuge for 3 minutes. e. After centrifugation the tubes will show 3 different layers: i. Top : a column of plasma ii. Middle : a thin layer of white blood cells. iii. Bottom : a column of red blood cells. f. Hold the tube against the scale of the reading device so that the bottom of the column of red cells is aligned with the horizontal zero line. g. Move the tube across the scale until the line marked as 1.0 passes through the top of the plasma column. Make sure that the tube is vertical. h. The line that passes the top of the column of red cells gives the Haematocrit value.
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Automated Method Generation of electrical pulses (Hct). Passage of a cell through the of an impedance counter or through the bean of light in a light scattering instrument lead to the generation of an electrical impulse.
Particle
aperture
No. of pulses = count of particle of particle Average pulse Height = volume
Results Before the introduction of international system of unite (SI) units, the haematocrit (erythrocyte volume fraction = packed cell volume, PCV) was reported as a percentage rather than a decimal fraction e.g. PCV = 45%.
height
pulse
Summation of height = PCV
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In using International System of unit (SI), the results become PCV = 0.45 (Vol. Fraction). Expected Range Sex Male Female Children (5) years Infants (3 months) Newborn N.R. 0.4-0.54 0.4-0.54 0.38-0.44 0.35-0.40 0.44-0.64
9. Erythrocyte Sedimentation Rate (ESR) This is based on the fact that inflammatory processes cause an alteration in blood proteins, resulting in aggregation of red cells, which makes RBCs heavier and more likely to fall rapidly when placed in a special vertical tube. The faster the sedimentation rate, the higher the ESR value. ESR is a non-specific test, because abnormal results indicate a pathological state rather than a functional disturbance. Westergren Method
The Westergren method requires collecting 2 ml of venous blood into a tube containing 0 .5 ml of sodium citrate. It should
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be stored no longer than 2 hours at room temperature or 6 hours at 4C. The blood is drawn into a Westergren-Katz tube to the 200 mm mark. The tube is placed in a rack in a strictly vertical position for 1 hour at room temperature, at which time the distance from the lowest point of the surface meniscus to the upper limit of the red cell sediment is measured. The distance of fall of erythrocytes, expressed as millimeters in 1 hour, is the ESR. Wintrobe Method
The Wintrobe method is performed similarly except that the Wintrobe tube is smaller in diameter than the Westergren tube and only 100 mm long. EDTA anticoagulated blood without extra diluent is drawn into the tube, and the rate of fall of red blood cells is measured in millimeters after 1 hour. The shorter column makes this method less sensitive than the Westergren method because the maximal possible abnormal value is lower. However, this method is more practical for demonstration purposes. Principle The red cells settle to the bottom of a long graded tube held in a vertical position leaving a layer of plasma above. The height of the column of plasma after 1 hour indicates the sedimentation rate of the erythrocytes (red cells).
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Reagents Trisodium Citrate 3.8% Trisododium Citrate anhydrous Distilled water Mix and keep in the refrigerator Procedure 1. Add 1.6ml of whole blood or collected with K2 EDTA to 0.4ml of the trisodium citrate solution. 2. Mix and draw the citrated blood into the westergren tube up to the 0-mark. 3. Place the tube in the Westergren stand in an upright position. 4. Wait for one hour. 5. Read the height of the plasma in mm starting from the 0-mark. The ESR increases with temperature above 23C. Use the following chart for correction of ESR reading (Figure 9.1). 3.8g 100ml
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Temperature
Figure 9.1 Reference values Sex Male Female N.R. 1 10mm/hr 3 15mm/hr
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The following table demonstrates some conditions accompanied by increased in the ESR level Rheumatic fever Rheumatic arthritis Coronary thrombosis Pneumonia Nephritis Cancer Multiple myeloma Nephrosis Metallic poisoning Syphilis Tuberculosis Anemia Leukemia Pregnancy Menstruation Agranulocytosis
Sources of Error in the Sedimentation Rate a. Unclean sedimentation rate tubes: Dirt, water, alcohol, ether, etc., will cause hemolysis and decrease the sedimentation rate. b. Partially clotted blood will decrease the sedimentation rate. c. Old blood: the blood must be fresh; therefore it must be used within 2 hours after withdrawal. As the blood stands, the red cells becomes more spherical and thus less inclined to assume rouleau formation. This decrease in rouleau formation decreases the sedimentation rate. d. Failure to mix blood: The test tube containing the blood should be completely inverted 10 to 12 times before filling the sedimentation rate tube. e. Use of cold unmixed blood. f. Air bubbles in the column of blood.
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g. Inclined sedimentation rate tube. The sedimentation rate tube must be placed in an exact vertical position.
10. Sickle Cell Test The purpose of this test is to detect sickle cell disorder (Anemia or Triat). Sickle cell anemia is caused by an abnormal form of hemoglobin known as hemoglobin-S, which tends to precipitate in such a way that the red cell takes the sickling shape. Principle One drop of blood is mixed with one drop of a sodium metabisulfite reagent on a slide. If the red cells contain abnormal hemoglobin, they will become sickle-shaped (or half-moon shape). The reagent sodium metabisulfite removes oxygen from the cells, allowing sickling to take place. Reagents Sodium metabisulfite 2% Aqueous Solution: Sodium metabisulfite (Na2S2O5) 0.2g Distilled water 10ml Always should be freshly prepared before use.
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Procedure a. Place a small drop of capillary blood in the centre of a slide. b. Add an equal drop of the fresh sodium metabisulfite solution. c. Mix carefully and cover with a cover slide, make sure that no air bubbles form. Seal with Vaseline or DPX. d. Place the slide in a wet chamber. e. Examine under the microscope after 15 minutes using the X40 objective. If the result is negative, re-examine after one hour, then after a further hour and after 24 hours. f. The test is negative if the red cells remain round. g. The test is positive if the cells become sickle-shaped, or banana-shaped, often with spikes.
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11. Coagulation 11.1 Bleeding Time The bleeding time (BT) is the interval between formation of a controlled, standard wound and the time the wound stops bleeding. The BT is prolonged in disorders of platelet function or capillary integrity. Specimen Free flowing capillary blood from the extensor (volar) surface of the forearm is collected on clean filter paper. Patient must avoid aspirin or acetyl-salicylic acid containing drugs for one week prior to test. Materials and Equipment 1. 2. 3. 4. 5. Simplate or Surgicutt (ET lancet device) Sphygmomanorneter Interval timer (stop watch) Filter paper Butterfly bandage
Procedure 1. Select a site on the extensor (valor) surface of the patients forearm distal, to the antecubital fossa. Avoid surface veins, scars, and bruises. If the forearm is hairy, lightly shave the area. Clean with alcohol and allow air-drying.
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2. Place the sphygmomanometer cuff on the upper arm and inflate to 40 mm Hg. The time between inflation of the cuff and incision should be 30-60 seconds. Ensure the pressure remains constant throughout the procedure. 3. Remove the bleeding time lancet from the pack and prepare for incision according to the manufactur ers instructions. There is always an arming device or safety to be activated before triggering. 4. Place the device firmly on the selected site on the forearm but do not press down. The incision should be made parallel to the fold of the elbow, across the tong axis of the arm. The length and depth of the lancet incision must be uniform from patient to patient. For this reason, no pressure is applied at the time of triggering the device. 5. Depress the trigger and simultaneously start the timer. Remove the bleeding time lancet about 1 second after triggering. 6. At 30 seconds, blot the flow of blood with the edge of the filter paper. Do not touch the edge of the wound with the paper. 7. Continue to blot every 30 seconds until blood no longer stains the paper. Stop the timer. 8. Remove the sphygmomanometer cuff, clean the arm, and apply a butterfly bandage across the incision, drawing the
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edges of the wound together. Advise the patient to keep the bandage in place for 24 hours. Reporting Results 1. Record results to the nearest 0.5 minutes. 2. Normal range is 2.5-9.5 minutes. Clinical Significance Prolonged in thrombocytopenia when the platelet count is below 100 X 109\l. Prolonged after aspirin ingestion. Prolonged in qualitative platelet disorders such as Glanzmanns thrombasthenia, storage pool disorder, or aspirin- like syndrome. The BT is routinely performed as a pre-surgical screen.
Limitations The bleeding time (BT) is actually a qualitative test indicating the presence or absence of abnormality. A prolonged BT test must be followed up with platelet count and aggregometry.
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11.2 Prothrombin Time (PT) Manual Method The Prothrombin Time (PT) is the time required for plasma to clot after tissue thromboplastin and optimal amount of calcium chloride have been added. The PT is a measure of extrinsic clotting pathway. Principle One stage Prothrombin Time (PT) The PT is the time required to form a fibrin clot when plasma is added to thromboplastin calcium mixture. The test is a measure of the extrinsic pathway of coagulation involving factors II, V, VII, IX and X (as well as fibrinogen). Tissue thromboplastin activates factor VII, proceeds through the cascade, ultimately generating thrombin. The thrombin thus form converts fibrinogen to fibrin. The rate of fibrin formation therefore depends on the level of factor II, V, VII, and X, and fibrinogen, and thus measures the overall activity of these factors. The test is used as screening procedure to indicate possible factor deficiency of extrinsic pathway.
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The PT test is sensitive to vitamin K dependent factors of the extrinsic pathway (factors II, VII, and X) and therefore is used as a means of monitoring oral anticoagulant therapy. Procedure 1. Place a small amount of patient plasmas and controls to be tested into tubes and Incubate in 37C water bath for 5 minutes. 2. Place 0.2 ml of thromboplastin reagent into a series of tube (2 X the number of tests to be performed), and incubate in 37C water bath for 3 minutes. 3. Forcibly deliver 0.1 ml of plasma to be tested into a tube containing the pre-warmed thromboplastin reagent, and simultaneously start a stopwatch. 4. Then begin swirling the tube in the water bath until 10 seconds have elapsed. Quickly remove tube from the water bath and wipe off. 5. Gently tilt the tube back and forth and stop the stopwatch at the earliest appearance of fibrin strands. 6. Report PT to the nearest tenth of a second. 7. Duplicate determinations should be done on each specimen and the results should agree within 0.6 seconds.
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Interpretation Normal Prothrombin Time (PT) is 12 to 14 seconds The preferred method to reports the patients PT in seconds to compare it with normal control time also reported in seconds. 11.3 Activated Partial Thromboplastin Time (aPTT) Principle The time required for plasma to clot after a measured amount of phospholipids extract from brain or lung called partial thromboplastin and CaCI is added. The interval is prolonged when any coagulation factor of the intrinsic or common pathway is deficient. The test is not sensitive to moderate reductions in levels of fibrinogen . Manual Method 1. Test plasma collected in sodium citrate (3.2% or 3.9%) as an anticoagulant from whole blood, assayed or frozen less than four hours after collection. 2. Control plasma, reconstituted according to manufacturers instructions.
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3. Partial thromboplastin reagent, reconstituted according to manufacturers directions. Reconstitute only enough for one days run and store at room temperature. 4. 0.025 M CaCI. 5. Water incubator set at 37 +/-0.5C. 6. 0.1 ml and 0.2 ml pipits, tips, 12X75 mm glass tubes. 7. Interval timer. Procedure 1. Place enough 0.025 M CaCI for the entire test run into a test tube and incubate at 37C for at least five minutes. Reagent may be incubated for several hours provided there is no evaporation. 2. For each test or control plasma to be assayed, pipette 0.1 ml partial thromboplastin reagent into a labeled test tube and place in water bath for three minutes. 3. Add 0.1 ml test or control plasma into the tube with partial thromboplastin, mix and incubate for exactly three minutes. 4. When reagent and plasmas are correctly warmed, forcibly add 0.1 ml CaCl to the mixture and simultaneously start the timer.
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5. Immediately begin swirling the tube in the water bath until 20 seconds have elapsed. Remove the tube from the bath and wipe. 6. Gently tilt the tube back and forth. Stop the timer at the first appearance of fibrin in the plasma. Record the results to the nearest tenth of a second. 7. Perform steps 2-6 for each determination. 8. Duplicate determinations are required for each test or control plasma and results must agree within seconds. The average of two determinations is the actual lest result. Expected Results Normal control is between 25-35 seconds. Significance 1. Prolonged in congenital or acquired single or multiple intrinsic or common factors deficiency. 2. Used to test for effectiveness of heparin therapy. 3. Prolonged in deficiency of factors (II, V, VIII, IX, X, XI, XII, HMWK, kallikrein, or when fibrinogen levels are below 100 mg/dl).
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4. Prolonged in circulating inhibitor, vitamin K deficiency, intestinal mal-absorption, liver disease, or obstructive jaundice. 11.4 New Methods: Semi-Automated and Full Automated Techniques Automated Methods Optical or Spectro-photometric Principles a. Photo-optical principle Optical systems are based on the notion that clot formation induces change in the plasmas optical density. As the clot is formed, there are changes in the optical characteristics from the initial reading of the plasma/reagents. These changes are monitored and used to derive the time taken for a particular degree of change to occur. b. Nephelometric principle The nephelometric principle is employed by some systems. In coagulation assays, a monochromatic laser light source is transmitted for example, by fiber optics. The light dispersion readings are made possible by a sensor that may be installed at 90 or 180 degrees from the light path, depending on the particular system, which then measures scattered light at an angle or records the change in light transmission. When the light reaches insoluble complexes
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such as fibrin fibers, it disperses in forward scattered angles (180 degrees) and lateral scattered angles (90 degrees). The chronometer stops when the amount of scattered light or transmitted light reaches a specific predetermined level. The difference between light scattered or transmitted before and after the clot formation is normally proportional to the amount of fibrin formed. c. Chromogenic principle This is based on the use of a color-specific generating substance known as chromophore, of which paranitroaniline (pNA) is the most common. It has a maximum absorbance at 405 nm. The principle of chromogenic testing resides in adherence of pNA to synthetic substrates. pNA is attached to a series of amino acids that mimics the target sequence of the activated coagulation factors needed to be determined. The coagulation protein cleaves the chromogenic substrate at a specific site between a defined amino acid sequences and releases the pNA. The intensity of the yellow color is proportional to the amount of pNA released. This is can be measured by photo detection at 405 nm wavelength. As more pNA is cleaved and freed, the absorbance capacity of the sample increases, which leads to greater change in the solutions optical density.
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d. Immunological principle Latex microparticles coated with a specific antibody are generally used against the analyte (antigen) being measured. A beam of monochromatic light goes through a latex microparticle suspension. When the wavelength is greater than the suspension particle diameter, the particles absorb a small amount of light. Yet, when the specific antigen-coated latex microparticles come in contact with the antigen present in the solution, they adhere to the antibody, forming links between the particles, which produces agglutination. When the particles diameter approaches the wavelength of the monochromatic light beam, a greater amount of light is absorbed. This increase in light absorbance is proportional to the agglutination, which, in turn, is proportional to the amount of the antigen present in the sample. This type of technology is available in more sophisticated coagulation analysers introduced in the market in the 1990s. Usually time-consuming standard immunological assays can be performed in minutes when using any of these automated tools. Advantages of Automation in a Coagulation Laboratory 1. Improves the capacity and flexibility of professional time spent. 2. Improves test reproduction. In the past, manual coagulation tests were inaccurate, with variation coefficients greater than 20%; the semi-automatic
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equipment provided greater accuracy in coagulation testing. However, with manual dispatch of samples and reagents, testing has to be done in duplicate. With totally automated equipment accuracy improved, attaining variation coefficients of less than 5%, and even 1% for some tests. 3. Reduces cost in samples and reagents. 4. Facilitates data storage and recovery systems by means of computer programs. 5. Allows automatic replay of results when mistakes are made in the first run. 6. Offers the possibility of running different tests using a single sample. 7. Permits sampling from a closed tube, which improves safety and efficiency in coagulation tests. This reduces, to a great extent, the possibility of exposing the operator to sprays or patient sample spills, or mistakes in labeling. 8. Provides capacity to dilute samples, calibrators, and controls. The equipment can be programmed for additional dilutions if the initial results escape the methods linearity. It can also autom atically carry out other tests without the operators intervention if clinically indicated or because of initial run results.
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9. Most analyzers include alarm systems that warn the operator of excess in pre-established readings, which may identify equipment problem (e.g. small amount of reagent, temperature failure, low sample level, and quality control errors). The different methodological types available have advantages and disadvantages that should be known and understood in order to guarantee precision and validity of test results. It is important to consider that laboratories are responsible for trustworthy results. A laboratorys main concern is to select the coagulation equipment that will generate appropriate results in spite of budget restraints. Such instruments demand regular technical maintenance, permanent knowledge, and system control, since a mistake or failure may decisively influence a number of results. Control systems that guarantee analytical confidence are therefore compulsory. Many laboratories may be fortunate enough to be able to evaluate equipment before purchasing. If this is not possible, it is very important to obtain adequate information and advice from a reference laboratory. When evaluating new equipment before purchase, first compare analyzers according to certain criteria such as: Equipment cost. Inactivity period and reliability. Repair response time. Ease of use.
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Availability of adequate maintenance within an appropriate timeframe. Validation process and throughput. Cost of disposable elements. Flexibility in using reagents from other manufacturers. Possibility of adding new tests protocols. Training courses and continuous training support.
Table 11.1: Explains the advantages and disadvantages of different detection methods in defining parameters.
Method Mechanical Advantages No interference due to physical characteristics such as lipemia or hemolysis. Use of small sample volumes. Whole blood can be used to eliminate centrifugation step. Possibility of graphics on clot formation. Optical checks for hemolysis/lipemia/icterus on some optical systems. Disadvantages Impossible to observe graphics of clot formation. May present problems of endopoint detection in some samples with low fibrinogen. Interference due to lipemia, hemolysis, hyperbilirubinemia, or protein increase on some systems. Some systems may present difficulties with clot detection when using some completely transparent reagents.
Photo-optic
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Contd.: Method Advantages Disadvantages Very short coagulation periods may go undetected owing to delay prior to initiation of monitoring. Limited number of available tests. Expensive costs of reagents. Limited by the instruments wavelength. Requires large test volumes for positive costbenefit ratio. Cost of instrument and reagents.
Nephelometric
Measurement of residual amount of Ag-Ab reaction. Fully specific assays may be easier. Additional parameters not suitable for measurement by clot detection may be possible. Increases the list of possible tests. Possible improvements in precision compared to clot based analyses. Fully automated analyzer reduces time for the test. Increases the number of possible tests.
Chromogenic
Immunological
Limited number of tests available. Cost of instruments. Cost of reagents.
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12. ABO and Rh-Grouping 12.1: History Landsteiner had discovered the ABO Blood Group System in 1901. He and five co-workers began mixing each others red blood cells and serum together and accidentally performed the first forward and reverse ABO groupings. Landsteiner's rule: If an antigen (Ag) is present on patient's red blood cells, the corresponding antibody (Ab) will be absent in the patient's plasma, under normal conditions. 12.2: Blood Group System Is a series of antigens exhibiting similar serological and physiological characteristics, and inherited according to a specific pattern. 12.2.1: Importance of ABO There are two Principles 1. Almost all normal healthy individuals above 3-6 months of age have naturally occurring antibodies to the ABO antigens that they lack (Table 12.1). These antibodies (Abs) termed naturally occurring because they were thought to arise without antigenic stimulation.
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Table 12.1: Illustrates the different blood groups and the Ags present in each, also demonstrates the Abs which present in each blood group.
ABO Group A B O AB Antigen Present A B NONE A and B Antigen Missing B A A and B None Antibody Present Anti-B Anti-A Anti-A&B None
2. These naturally occurring Abs are mostly IgM class. This means that, they are Abs capable of agglutinating saline/low protein suspended red cell without enhancement and may activate complement cascade. Therefore, ABO compatibility between donor and recipient is crucial since these strong, naturally occurring A and B antibodies are IgM and can readily activate complement and cause agglutination. If ABO antibodies react with antigens in vivo, the result is acute hemolysis and possibly death. 12.2.2: Indications for ABO grouping ABO grouping is required for all of the following individuals: blood donors, transfusion recipients, prenatal patients, newborns and paternity testing.
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12.3: ABO Typing ABO typing involves both antigen typing and antibody detection. The antigen typing is referred to as the forward typing (cell) and the antibody detection is the reverse typing (serum). 1. Forward typing: determines antigens (Ags) on patient's or donor's cells. Cells are tested with the different types of antisera reagents such as anti-A, anti-B, (and in the case of donor cells anti-AB).
2. Reverse typing: determines antibodies (Abs) in patients or donor's serum or plasma is also known as back typing or serum confirmation. Serum tested with reagent A1 cells and B cells.
12.4: Principle and Applications The ABO system is the most clinically significant blood group system for transfusion practice, because it is the only blood group system in which antibodies are consistently and predictably present in the serum of normal individuals whose red cells lack the antigen. ABO compatibility
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between donor and recipient is the foundation upon which all other pre-transfusion testing rests. The D (Rho) antigen is consider as one of the most immunogenic, after A and B antigens, the most important red cell antigen in transfusion practice due to its potent antigenicity. Unlike the ABO system, however, individuals who lack the D antigen do not consistently and predictably have anti-D in their serum. The ABO and Rh are determined for all patients who are candidates for transfusion, for all blood donors, for all prenatal patients, and for all potential organ recipients and donors. Methodology 1: Slide Method The slide method is less satisfactory than the tube method. Agglutination is rapid on that slide; this method is useful when only one or two samples of blood are to be tested. Grouping anti-sera should be obtained ready for use from a known source and should be stored according to the recommendations of the manufacturer. They must be kept free from bacterial growth to avoid non-specific falsepositive results.
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Procedure 1. Put one drop of the patients whole blood to each labeled square on a special slide (A, B, AB and D). 2. Add one drop of each grouping anti-sera (anti-A, anti-B, anti-AB and anti-D), respectively. 3. Mix the blood and the anti-sera in each square with a wooden-stick. 4. The results may be read within 1-5 minutes. 5. Presence of agglutination means a positive reaction. 6. Doubtful presence or absence of agglutination may be checked by viewing under the microscope. 2: Tube Method 4-5 ml clotted blood from an adult or a 2 ml clotted blood from a pediatric patient should be collected for ABO reverse grouping and the same volume of blood in EDTA tube or citrate phosphate dextrose adenine (CPDA) (CPDA1) for forward grouping. ABO forward grouping may also be done on a sample from a segment on a donor unit.
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Reagent and Equipment Reagent (Anti-sera) Anti-A, Anti-B and Anti-AB, Anti-D Reagent (Cells) A1, A2 and B Cells Tubes 12 x 75 mm and test tube rack Marking pen Disposal pipettes Physiological saline Centrifuge Lighted agglutination viewer
Preparation of Washed Cell Suspensions 2-5% Red Blood Cell Concentration in Saline Between 2-5% cell suspension provides optimum antigen concentration in the tube method for red blood cells typing.
Washing Red Blood Cells The aim of washing the red blood cells is to remove plasma, which contains substance that may interfere with antigenantibody reaction. The following may be in the plasma and may interfere with testing: Interfering proteins such as Wharton's jelly that is seen in newborn cord blood, cold-acting autoimmune antibodies, and increased levels of immunoglobulins that may cause either agglutination or rouleaux formation.
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Fibrinogen can result in fibrin strands forming that makes grading reactions difficult.
Procedure 1. Place 1 to 3 drops of blood in the tube 2. Aim the tip of the saline bottle towards the center of the tube and forcibly squeeze saline into the tube. 3. Fill the tube 3/4 full of saline (there will be less splattering in the centrifuge) (Fig. 12.1). 4. Centrifuge for 60 seconds in calibrated centrifuge to pull most of cells into a button in the bottom of the tube. 5. Decant the saline completely. 6. Shake the tube to re-suspend cell button before washing the cells again. It will depend on the procedure being done as to how many washing steps are going to be done.
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Figure 12.1: Shows the Red Blood Cells Suspension in the Test Tube Washing Procedure.
Figure 12.2a: Demonstrates the different types of anti-sera (AntiB, Anti-B, Anti-Ab and Anti-D).
Figure 12.2b: Shows labeling of different tubes for reverse grouping.
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ABO Blood Group Blood Grouping Procedure Confirm that patient information on the sample matches the information on the worksheet. 1. Centrifuge the samples and remove the serum to a labeled tube. 2. Label 8 tubes with the first 3 or 4 letters of the patients last name/or first 3 or 4 digits of identification number and line them up in test tube rack. 3. Add 2-3 drops of the patient's cells to the washed cells tube, and prepare a washed suspension of 2-5%. Add 2 drops of patient's serum to both A1 and B tubes. 4. Add one drop of reagent anti-A to the A tube, one drop of reagent anti-B to the B tube, and one drop of reagent anti-A, B to the A, B tube (Figure. 12.2a). 5. Add one drop of anti-D reagent to the D tube. 6. Add one drop of patient washed cells to the following tubes: A tube B tube AB tube D tube. 7. Add one drop of reagent A1 cells to the tube which has been labeled as A1 tube, and one drop of B cells to the tube which has been labeled as B tube (Figure. 12.2b).
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8. Shake all 6 tubes gently to mix. 9. Centrifuge tubes in the centrifuge for the calibrated time for saline suspended cells. 10. Gently re-suspend each cell button individually and examine for hemolysis or agglutination with the aid of the lighted agglutination viewer. Grade results negative (0) to positive (4+). Immediately record the results in the appropriate spaces on the worksheet. 11. Discard all materials in the isolation trash containers. Results Positive Agglutination (+): The red cells form one or more clumps with a clear supernatant fluid. Negative Agglutination (0): The red cells re-suspend easily without any visible clumps.
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Grading System: Table 12.2: Explains the different types of agglutination reaction between Ag & Ab in blood grouping. Solid clumping indicated by 4+ and no agglutination indicated by (0) as seen in figure 12.3. 4+ 3+ 2+ 1+ 0 Solid clump. Several large clumps. Small to medium sized clumps; clear background. Small clumps; cloudy background. Negative: the red cells resuspend easily without any visible clumps.
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Figure 12.3: Illustrates the different agglutination reaction strength between Ag & Ab either for forward and reverse grouping.
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Interpretation The following Tables (12.3a): Demonstrates the reaction between Ag & Ab in forward grouping using patients RBC with anti-sera. Positive with anti-A (4+) indicated that the patients blood grouping is A. Positive with anti-B (4+) indicated that the patients blood is group B.
ABO Blood Group A B O AB Patient Red Cells Tested with Known Anti-sera Forward Grouping Anti-A Anti-B Anti-A,B 4+ 0 4+ 0 0 4+ 4+ 0 4+ 4+ 0 4+
+ = Agglutination (graded 1+ to 4+); 0 = No agglutination
(12.3b): Demonstrates the reaction between patients serum and known cells in the reverse grouping positivity indicated the presence of Ag & Ab reaction (4+) and negativity by (0), there is no Ag & Ab reaction.
ABO Blood Group A B O AB Patient Serum Tested with Known Reagent Cells Reverse Grouping A Cells B Cells 0 4+ 4+ 0 4+ 4+ 0 0
+ = Agglutination (graded 1+ to 4+); 0 = No agglutination or hemolysis
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Technical Errors: The first step in resolving a discrepancy is a careful repeat of the entire test procedure, paying careful attention to details possible pitfalls are list below: a. Partial drying of the slide in cell groupings may be misinterpreted as agglutination. b. Over centrifugation or under centrifugation may result in false positive or false negative readings respectively. c. Rough dislodgement of the centrifuged cell button may disrupt small agglutination and result in false negative reading. d. The cell-serum mixture may be accidentally heated, resulting in false negative reading. e. The use of improper concentration of cells or old cells may lead to a false negative reading. f. Failure to observe haemolysis will result in false negative readings. g. Dirty glassware will stimulate clumping and result in a false positive reading. h. The specimen may be identified incorrectly.
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13.
Anti-Human Globulin (AHG) Types of AHG There are two different types of anti-human globulin test (AHG): 1. Indirect of anti-human globulin test known as (IDAT, IYT) test which formed in vitro. 2. Direct anti-human globulin test known as (DAT) test which formed in vivo.
The direct anti-human globulin test does not require the incubation phase, because the antigenantibody complexes have formed in vivo. For indirect anti-human globulin test (in vitro) antigenantibody reactions the following is required; 1. Incubate red cells with anti-sera to allow time for antibody molecules attachment to red cells antigen. 2. Perform three times saline washing to remove free globulin molecules. 3. Add anti-human agglutinates. globulin reagent to red cell
4. Centrifuge, to accelerate agglutination by bringing cells closer together.

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5. Examine for agglutination to interpret test as positive or negative. 6. Grade agglutination to determine the strength of the reaction. 7. Add antibody coated red blood cells to those with negative reaction, to checks for neutralization of antihuman sera by free globulin molecules (Coombs control cells are D-positive red blood cells that are coated with anti-D). Indirect AHG Test Applications
There are many applications for this test including; 1. Detection of incomplete antibodies to potential donor red cells (compatibility testing) or to screening cell (antibody screen) in serum. 2. Identification of antibody specificity using a panel of red cells with known antigen proteins. 3. Determination of red cell phenotype using known antisera for example (Kell typing, Du testing). 4. Titration of incomplete antibodies.
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Principle and Applications of (DAT) Direct anti-human globulin test is used to detect in vivo sensitization of red blood cells. Clinical conditions that can result in vivo coating of red blood cells with antibody or complement or both are; 1. Hemolytic disease of the new born (HDN). 2. Hemolytic transfusion reaction (HTR). 3. Auto-immune and drug-induced hemolytic anemia.
Direct anti-human globulin Testing Panel (DAT)
Initial (DAT) should include both anti-IgG and anti-C3d reagents or poly-specific (anti-IgG-C3d) reagent. A DAT panel using poly-specific and mono-specific reagents characterizes the type of protein sensitization that has occurred in vivo. A DAT panel is useful in determining whether the patients red cells are coated with antibody, complement, or both. Procedure 1. Prepare a 2-5% cell suspension in normal saline for the cells to be tested.
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2. Add 2 drops of the prepared cell suspension to a tube marked test and 2 drops to a tube marked auto control. 3. Wash cells 3 times by filling both tubes with fresh normal saline and centrifuging according to calibration of centrifuge for cells washing. Decant supernatant completely after each washing (Cord blood specimens must be washed a minimum of six times). 4. After the third washing, add two drops of Coombs antihuman globulin (AHG) reagent to the dry cell button in the tube marked test. Do not re-suspend the cells in the test tube with saline before adding the Coombs serum or reagent. 5. Add two drops of saline to the tube marked auto control. 6. Re-suspend the cell buttons in both tubes with gentle shaking and centrifuge according to calibration of centrifuge for the antihuman globulin (AHG) technique. 7. Read macroscopically by holding the tubes to a light source and gently a agitating them. Examine for agglutination. Read microscopically only if results are macroscopically doubtful. 8. Cared the reaction from 4+ to 0.
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Antibody Detection and Identification Purpose The purpose of the antibody screen is to detect red blood cell antibodies other than anti-A or anti-B. These antibodies are called unexpected because only 0.3 to 2 % of the general population have positive antibody screen. Once an unexpected antibody is detected, antibody identification studies are performed to determine the antibodies specificity and clinical significance. All red blood cell antibodies are significance if they cause shortened survival of antigen positive red blood cells. For example, anti-D is a clinically significant antibody, because it will bind to D-positive red blood cells, resulting in immune destruction or hemolysis. Therefore, proper detection and identification of red blood cell antibodies is important for the selection of appropriate blood for transfusion and in the investigation of hemolytic disease of the new born and immune hemolytic anemia. Antibody screening test involve testing patients serum against two or three reagent red blood cell samples called screening cells. Screening cells are commercially prepared group O cell suspensions obtained from individual donors that are phenotype for the most commonly encountered and clinically important red blood cell antigens.
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Group O cells are used so that naturally occurring anti-A or anti-B will not interfere with detection of unexpected antibodies. The cells are selected so that the following antigens are present on at least one of the cell sample: D, C, E, c, e, M, N, S, s, P, Lea, Leb, K, k, Fya, Fyb, and Jkb An anti-gram listing the antigen makeup of each cell provided with each lot of screening cells issued from a manufacture (Fig. 13.3). It is important that the lot number on the screening cells matches the lot number printed-on the anti-gram because antigen make up will vary with each lot.
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Figure 13.3 Explanation of an anti-gram listing the antigen makeup of each cell provided with each lot of screening cells issued from a manufacture
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Screening Cells The screening cells are available in three different forms: 1. A single vial of no more than two donors pooled together in one vial. 2. Two vials each with different donors. 3. Three vials representing three different donors. Two or three cells screening sets are required for detection of antibodies in pre-transfusion testing. Auto-logous Control Autologous control is considered as part of the Ab screening; it can be performed in parallel with the Ab screen and involves testing the patients serum against the patients red blood cells. A positive auto-logous control is an abnormal finding and usually means that patient has a positive direct anti-globulin test (DAT). Procedure 1. Label three test tubes as 1, 2 and 3. 2. Add 2 drops of patients serum to each tube.
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3. To tube No. 1, add two drops of screening cells No. 1. To tube No. 2, add two drops of screening cells No. 2. To tube No. 3, add two drops of screening cells No. 3. 4. Spin; read macroscopically this is considered as immediate spin (IS). 5. Incubate at 37C for 30 60 minutes 6. Spin, read macroscopically, if no agglutination, go to next step. 7. Wash all the tubes 3 times with normal saline. 8. Add to each tube 2 drops of Anti-human globulin (AHG) reagent. 9. Spin, read macroscopically and microscopically. Any agglutination or haemolysis detected at any step, means indirect Coombs is positive. 10. If the test was negative then a control cell (Check Cell) must be added and re-centrifuge all the tubes. 11. Read all the tubes macroscopically and microscopically observe for agglutination. If there is positive agglutination that indicates the final reading is valid. However, if the results were negative indicates that the test results were invalid and the test must be repeated again.
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Interpretation Evaluation of the antibody screening and auto-logous control results can provide clues and give direction for the identification and resolution of the Ab or Abs. The investigator should consider the following questions. In what phase (s) did the reaction (s) occur? Low temperature: Abs of the IgM class will react best at low temperatures and are capable of causing agglutination of saline-suspended red blood cells (immediate spin reading (IS). Of the commonly encountered Abs are, anti-N, -I. and -P (IgM). Abs of the IgG class (warm Abs) will react best at the AHG phase (37C). The most common Abs are: anti-Rh and ante- kell.
Is the auto-logous control negative or positive? A positive Ab screen and a negative auto-logous control indicate an allo-antibodv has been detected. A positive auto-logus control may indicate the presence of autoantibodies, antibody to medication or it may idiopathic, if the patient has been recently transfused, the positive autologous control may be due to alloantibody coating circulating donor red blood cells.
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Did more than one Ab screening cell react? If the patient has multiple Abs, when the Abs corresponding Ag is found on more than one screening cell, or when the patients serum contains an autoantibody, more than one screening cell will be positive.
Did the Ab screening cells react at the same strength and phase? A single Ab specificity should be suspected when all cells react at the same phase and strength. Multiple Abs is most likely when cells react at different phases and strengths and auto-antibodies are suspected when the auto-logous control is positive.
Coombs Cells (C.C) Control cell used when AHG test is negative. To confirm that washing has been adequate and the anti-globulin reagent is reactive. They can be used to ensure that AHG test with negative results are not false negative because of inactivation of the AHG reagent. When AHG test is negative, they should be free AHG reagent in the test tube. When the CC is added, the free AHG in the test should cause agglutination of the sensitized RBCs.
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Preparation of Control Cell A group O Rh positive donors sample is mixed with 1:10 dilution of reagent anti-D and allowed to incubate. After sensitization of RBC, they are washed with saline and suspended to a 50% RBC suspension. These sensitized RBCs then used to confirm the anti-IgG activity of the AHG.
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Atlas of Peripheral Blood Smears
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Normal erythrocyte: Normal erythrocyte Is a mature non-nucleated red cell appearing as a biconcave disc. It should stain pink to red with a central pallor occupying 1/3 the diameter of the cell with a Wright-Giemsa stain. Figure XIII. 1 Normal Erythrocyte
Echinocytes (Burr Cells, Crenated Cells) Echinocytes are normochromic red cells with blunt short. projections, which are evenly distributed over the surface of the red blood cell. Echinocytes are commonly seen in: Renal disease Liver disease
Figure XIII. 2 Echinocytes
Macrocytes Macrocytes are large red cells with a high mean corpuscular volume (MCV), usually greater than 100 fl. Their hemoglobin concentration is normal. They may be oval or round. Macrocytes are commonly seen in: Normal newborn Drug associated macrocytosis (e.g., anticonvulsants, antidepressants, sulpha, chemotherapeutic agents, estrogen and antiretroviral agents) Folate deficiency BI2 deficiency Dyserythropoiesis Liver disease
Figure XIII. 3 Macrocytes
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Microcytes Microcytes are smaller than normal red cells with a MCV less than 75 fl in children less than 5 years of age and less than 80 fl in children over 5 years of age. Microcytosis is usually associated with hypochromia. Microcytic hypochromic cells are commonly seen in: Iron deficiency anemia, Lead poisoning Figure XIII.4 Microcytes
Ovalocytes (Elliptocytes) Ovalocytes and elliptocytes are red cells that are elongated with blunt ends and parallel sides. The term ovalocyte is interchangeable with the term elliptocyte. Their names are descriptive of their appearance. A small number of elliptocytes are seen in the normal peripheral smear. elliptocytes are commonly seen in: Hereditary elliptocytosis Renal and liver diseases Vitamin B12 deficiency Myelodysplasia
Figure XIII.5 Ovalocytes and elliptocytes
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Polychromatophilic Red Cells (Reticulocytes) A polychromatophilic red cell is a non-nucleated red cell precursor slightly larger than the mature red cell (8-10 microns in diameter). It contains RNA in addition To the hemoglobin and stains gray blue or pale purple with Wright-Giemsa stain. It has a deep blue granular or filamentous structure when supravitally stained. Reticulocytes are seen in: Hemolytic anemias Blood loss anemias Recovering anemia Figure XIII.6 Reticulocytes
Sickle Cells (Drepanocytes) Sickle cells are red cells with two pointed ends which are in the shape of a crescent or sickle. This is due to the polymerization of deoxygenated hemoglobin S causing changes to the red blood cell making it less deformable and much more rigid. Sickle cells are usually seen in: Sickle cell anemia Hemoglobin SC Figure XIII.7 Sickle Cells
Spherocytes Spherocytes are dense, staining spherical red cells with normal or slightly reduced MCV without any central pallor. Spherocytes are commonly found in: Hereditary spherocytosis Autoimmune hemolytic anemia (warm antibody type) Post transfusion
Figure XIII.8 Spherocytes
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Stomatocytes Stomatocytes are red cells with a central clear opening appearing like a mouth, hence the name stoma, meaning mouth. Stomatocytes are commonly seen in: Hereditary Stomatocytosis Liver disease Figure XIII.9 Stomatocytes Target Cells (Codocytes) Target cells have a central hemoglobinized area within the surrounding area of pallor. These morphological features give these red cells the appearance of a sombrero or a bulls eye. Target cells are larger than normal cells with excess cell membrane. target cells are commonly seen in: Hemoglobin C Sickle cell disease Hemoglobin E Hemoglobin H disease Thalassemias Iron deficiency anemia Liver disease Figure XIII. 20 Target Cells
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Teardrop Cells (Dacrocytes) Red cells in the shape of a teardrop or a pear with a single short or long, blunted or rounded end are called teardrop cells. Teardrop cells are commonly seen in: Osteopetrosis Myelofibrosis Bone marrow infiltrated with hematological or nonhematological malignancies Iron deficiency anemia Pernicious anemia Anemia of renal disease
Figure XIII. 21 Teardrop Cells
Basophilic Stippling Basophilic stippling is a collection of fine or coarse granules in the red cells. Clinically insignificant, fine stippling is often seen in reticulocytes. Coarse stippling is seen in clinically significant conditions with impaired hemoglobin synthesis and is a result of accumulation of abnormal aggregates of ribosomes and polyribosomes. Basophilic stippling is commonly seen in: Lead poisoning Iron deficiency anemia Thalassemia Refractory anemia Figure XIII.22 Basophilic Stippling
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Hemoglobin C crystals: Hemoglobin C crystals are dense rhomboid, tetragonal or rod-shaped structures within red cells. They often distort the red cell and project beyond its rim. Hemoglobin C crystals are commonly seen in: Hgb CC Hgb SC
Figure XIII.23 Hemoglobin C crystals
Heinz Bodies Heinz bodies are multiple blue-purple inclusions attached to the inner surface of the red cell membrane. They are not visible in Wright-Giemsa-stained blood films, but are visible in supravitally stained smears. Heinz bodies are precipitated normal or unstable hemoglobin usually secondary to oxidant stress. Heinz bodies are commonly seen in: G6PD deficiency Unstable hemoglobins Congenital Heinz body (bite cell) anemias Howell-Jolly Bodies Howell-Jolly bodies are small round bodies composed of DNA, about 1 m in diameter, usually single and in the periphery of a red cell. They are readily visible on the Wright-Giemsa-stained smear. The spleen is responsible for the removal of nuclear material in the red cells, so in absence of a functional spleen, nuclear material is removed ineffectively. Figure XIII.25 Howell-Jolly Bodies Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE
Figure XIII.24 Heinz Bodies
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Howell-Jolly bodies are seen in: Post splenectomy Functional asplenia Anatomical absence of spleen Miscellaneous Abnormalities Agglutination Red cell agglutination occurs when red blood cells clump in irregular masses. Agglutination is secondary to cold agglutinins, most commonly an igm antibody. Red cell aggutination is most commonly seen in: Viral infections (e.g., influenza, parainfluenza) Lymphoproliferative disorders Plasma cell dyscrasias
F IGURE XIII.26 A GGLUTINATION
Rouleaux Rouleaux formation is a common artifact in the thick area of any blood film. True Rouleaux is seen in the thin part of the blood smear. There are four or more red cells organized in a linear arrangement like a stack of coins. The central pallor is generally apparent. True rouleaux formation is due to increased amounts of plasma proteins primarily fibrinogen and globulins. Rouleaux are commonly seen in: Infections Inflammation Monoclonal gammopathies Neoplastic diseases AIHA warm antibody disease F IGURE XIII.27 R OULEAUX
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Hemolytic Anemias in Pediatrics
Hereditary elliptocytosis Hereditary elliptocytosis (HE) is a common and mild anemia due to a structural defect of the erythrocyte membrane protein, spectrin. It is common in individuals of African and Mediterranean descent. Approximately 85% to 90% of patients have only morphological evidence of HE. The remainders of patients have a hemolytic anemia of varying severity. Spherocytic HE and stomatocytic HE (Melanesian or Southeast Asian Ovalocytosis) are described. Hereditary Pyropoikilocytosis is a related disorder.
Figure XIII.28 Hereditary elliptocytosis
Hereditary stomatocytosis Hereditary stomatocytosis (HST) is a mild autosomal dominant hemolytic anemia. There is an inherited abnormality in erythrocyte cation permeability, leading to abnormal erythrocyte hydration. The most common defect is in the red cell membrane protein, stomatin. F IGURE XIII.29 H EREDITARY
STOMATOCYTOSIS
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Hemoglobinopathies
Hereditary acanthocytosis Hereditary acanthocytosis is an autosomal recessive, mild hemolytic anemia due to a defect in beta lipoprotein.
Figure XIII.30 Hereditary acanthocytosis
Sickle hemoglobinopathies Sickle hemoglobinopathies are most commonly present in people of African descent. In the United States, these Pathologies are seen in African Americans, African immigrants, Caribbean and Central Americans, particularly from the Caribbean coast of Central America. In addition, sickle syndromes are seen in Middle Eastern, Mediterranean and East Indian populations. Hemoglobin S is a qualitative defect of the globin chain of the hemoglobin inwhich there is a substitution of amino acid valine for glutamic Acid leading to polymerization of hemoglobin in the presence of hypoxemia.
Figure XIII.31 Sickle hemoglobinopathies
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Dyskeratosis Congenita (DC) DC is a rare form of ectodermal dysplasia. The diagnostic triad consists of: Fifty percent of patients with DC develop aplastic anemia in the second decade of life and 10% develop cancer in the third and fourth decades of life.
F IGURE XIII.32 D YSKERATOSIS C ONGENITA (DC)
Myelodysplastic Syndrome (MDS) MDS is usually associated with pancytopenia and normocellular to hypocellular bone marrow. MDS is characterized by megaloblastic and dyserythropoietic erythropoiesis with maturational defect. Dysmyelopoiesis is common with maturational aberrations in myeloid cells. Megakaryopoiesis often is atypical. The common causes of MDS are: Drugs, toxins and chemicals Radiation Preleukemia Chromosomal syndromes of downs, other trisomies, monosomy 7 Refractory anemias F IGURE XIII.33 M YELODYSPLASTIC S YNDROME (MDS)
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Microcytic Anemias: Iron Deficiency Anemia (IDA) is the most common anemia worldwide and commonly affects infants between the ages of 9 months and 2 years, because of poor iron intake. Chronic blood loss is the commonest cause of IDA in children over 2 years of age.
Figure XIII.34 Microcytic Anemias
Lead Poisoning Anemia of lead intoxication is largely caused by inhibition of heme synthesis and inhibition of pyramidal 5' nucleosidase. Anemia of lead poisoning mimics IDA but has a normal iron profile. Basophilic stippling in the red blood cells and polychromatophilic cells are the hallmarks of lead poisoning. F IGURE XIII.35 L EAD P OISONING
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White Blood Cells (Leukocytes) Neutrophil, Segmented (Segs) The segmented neutrophil is the predominant white blood cell in the peripheral blood. It is 10 m to 15 m in diameter with pale pink cytoplasm and specific fine granules. Rare azurophilic (primary granules) are seen. The nucleus is lobulated (between 2 and 5 lobes) and the lobes are connected by a thin filament.
Figure XIII.36 Neutrophil,Seg. Band Neutrophil Band neutrophils constitute from 0% to 5% of the nucleated cells under normal conditions in the peripheral blood. The band is round to oval in shape and 10m to 18m in diameter. The nucleus can be band like, Sausage-shaped, S-, C- or U-shaped and may be twisted and folded on itself. The cytoplasm is pale with specific granules in it. Increased numbers of bands appear in the blood in a number of physiologic and pathologic states. Bands are increased in the peripheral blood in the following conditions: Severe infections--Sepsis/bacteremia Inflammation Stress
F IGURE XIII.37 B AND N EUTROPHIL
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Basophile In the normal physiological state there are very few (0%1%) basophils in the peripheral blood. All basophils, from the basophilic myelocyte to the mature segmented Basophil, are characterized by the presence of a moderate number of large coarse and densely stained granules of varying sizes and shapes. The granules in the WrightGiemsa-stained preparation are blue-black; Some may be purple to red. Basophils are increased in the blood in: Myeloproliferative disorders (e.g., chronic Myelogenous leukemia) Hypersensitivity reactions Mastocytosis
F IGURE XIII.38 B ASOPHILE
128
Eosinophil Eosinophils are distinct cells, about the size of a neutrophile (10 m -15 m) with abundant cytoplasm filled with many large, coarse, orange-red granules which are refractile because of their crystalline structure. About 80% of segmented eosinophils will have the classic twolobe appearance. Only 1% to 8% of circulating leukocytes are eosinophils. Morphologically abnormal eosinophils are seen in: Myelodysplastic syndrome Megaloblastic anemias Eosinophils are increased in the following conditions: Allergies Parasitic infestations Infections Acute leukemia Myeloproliferative diseases F IGURE XIII.39 E OSINOPHIL
129
Monocytes Monocytes are larger cells, 12 m to 20 m in diameter. The majority of monocytes are round with smooth edges. Usually, there is abundant gray to gray-blue cytoplasm which may contain fine, evenly distributed granules and vacuoles. The nucleus is usually indented, the chromatin is condensed and occasionally a small and inconspicuous nucleolus is seen. Monocytes are seen in 1% to 5% of the leukocytes in the peripheral smear. Monocytes are increased in the following conditions: Chronic infection (e.g., tuberculosis) Recovery from severe neutropenia in neoplastic or a plastic disorders Benign neutropenia F IGURE XIII.40 M ONOCYTES
131
Lymphocytes Most lymphocytes seen on a blood smear are fairly homogeneous. Lymphocytes are small, round- to avoid shaped cells that range in size from 7 m to 15 m with round to oval nuclei. Some normal lymphocytes are medium-sized due to an increase in the amount of cytoplasm. The nucleus appears dense or coarse and clumped with ridges of chromatin and parachromatin. nucleoli, if present, are small and inconspicuous. The majority of lymphocytes have a scant amount of pale blue to basophilic agranular cytoplasm. Reactive Lymphocytes Reactive lymphocytes are notable due to their remarkable heterogeneity. They tend to be large with abundant Cytoplasm. They are indented by surrounding red cells, and they may have blue skirting (blue outline around the Cytoplasm). Another descriptive term used is the fried egg appearance. This cell corresponds to the Downey Type II cell. Accompanying this type of reactive lymphocyte, plasmacytoid lymphocytes are frequently seen. These lymphocytes have deeply basophilic cytoplasm and resemble plasma cells. Their size varies from small to moderate and may have one or more prominent nucleoli. They correspond to Downey Type III cells. The Downey Type I cell which is not observed as frequently is small with a slightly basophilic cytoplasm and an indented or lobulated nucleus. Reactive lymphocytes are frequently seen in children with viral diseases, but the condition where reactive lymphocytes demonstrating all the downey type cells are seen is usually infectious mononucleosis.
F IGURE XIII.41 L YMPHOCYTES
F IGURE XIII.42 R EACTIVE L YMPHO .
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Toxic Vacuolization Large, clear areas in the cytoplasm of neutrophils
F IGURE XIII.43 T OXIC V ACUOLIZATION Hypersegmented Neutrophils Large hypersegmented neutrophils are a result of megaloblastic hematopoiesis. In megaloblastic myelopoiesis, eosinophils and basophils are large and also hypersegmented. To be considered hypersegmented, Neutrophils should have 6 or more lobes. Megaloblastic hematopoiesis is seen in: Vitamin B12 deficiency Folate deficiency
F IGURE XIII.44 H YPERSEGMENTED N EUTROPHILS
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Auer Rods Auer rods are pink or red, rod-shaped cytoplasmic inclusions seen in myeloid cells and occasionally in monocytes. Auer rods are thought to be an abnormal crystalline form of primary granules: Acute myeloid leukemia Myelodysplastic/pre-leukemic states. An immature myeloid cell containing multiple Auer rods clumped together is known as a faggot cell, and is seen in acute promyelocytic leukemia. Platelet Morphology: Platelets Normal Platelets (Thrombocytes) Platelets are small non-nucleated cells derived from the cytoplasmic fragments of megakaryocytes and are variable in size. Normalsized platelets are 1.5 m to 3 m in diameter and have fine purple-red granules aggregated at the center or dispersed throughout the cytoplasm: Normal platelets are 1.5 m to 3 m in diameter Large platelets are 4 m to 7 m in diameter Giant platelets are greater than 7 m in diameter and may be 10 m to 20 m. Small (micro) platelets are less than 1.5 m in diameter Figure XIII.46 Normal Platelets F IGURE XIII.45 A UER R ODS
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Large Platelets Large platelets are usually 4m to 7m in diameter. Large platelets are commonly seen in: Reactive thrombocytosis Autoimmune thrombocytopenia Myeloproliferative,disorder,leukemoid reaction Myelodysplastic disorder Neoplastic diseases: Acute Megakaryocytic Leukemia (M7) Hereditary thrombocytopenias
F IGURE XIII.47 L ARGE P LATELETS
Giant Platelets Giant platelets are larger than 7m and may be 10 m to 20 m in diameter. The periphery of the giant platelet may be round or scalloped. The cytoplasm may contain fine azurophilic granules or the granules may fuse into Giant forms. Giant platelets are commonly seen in: Myelodysplastic disorder Hereditary Thrombocytopenias, such as: May-Hegglin anomaly Storage pool syndrome F IGURE XIII.48 G IANT P LATELETS
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Small Platelets (Microthrombocytes) Microplatelets are usually less than 1.5 m in diameter and are not counted adequately by the impedance blood cell counters, giving spuriously low platelet counts. Microplatelets are seen inWiskott Aldridge Syndrome Figure XIII.49 Small Platelets
Platelet Satellitism Platelets sometimes clump and adhere to neutrophils and more rarely to monocytes forming platelet rosettes, which is known as platelet satellitism. Platelet satellitism is a cause of spurious thrombocytopenia because the cellular aggregates are counted as leukocytes rather than platelets. Quantitative Disorders of platelets: Thrombocytopenia Thrombocytosis Common Causes of Thrombocytopenia: Decreased production Aplastic anemia Acute leukemia Viral infections Parvovirus CMV Increased destruction Immune thrombocytopenia Idiopathic thrombocytopenic purpura (ITP)
F IGURE XIII.50 P LATELET S ATELLITISM
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Thrombocytosis Causes may include: Reactive thrombocytosis Post infection Inflammation Chronic diseases Juvenile rheumatoid arthritis (JRA) Collagen vascular diseases Benign tumors: adenomas, lipomas Ganglioneuroblastoma / neuroblastoma
Figure XIII.51 Thrombocytosis
Most of Reprinted Pictures from the DM96 machine Sysmex Middle Eastafter written permission (May 2011).
136
Appendix I
137
References 1. 2. UNRWA,(1999): Manual on Basic Techniques for UNRWA laboratory personnel, third edition). Keijzer M. and Der Meer W., (2002): Automated Counting Of Nucleated Red Blood Cells In Blood Samples Of Newborns,Clinical & Laboratory Haematology (24), 343 345. Briggs C. et al, (1999): The Automated Nucleated Red Cell Count On The Sysmex Xe-21oo, Department of Hematology, University College Hospital, Grafton Way, London, U.K. Murai M. et al, (1999): WBC Differential, Hematology, Complete Blood Count (CBC), Leukemia, Peripheral Blood Stem Cell (PBSC), Hematopoietic Progenitor Cell (HPC), Sysmex Journal, (2 ), 2, in Japanese. Buttarello, M. et al, (1996): Reticulocyte Quantification by Coulter MAXM VCS (Volume, conductivity, light scatter) technology, Clinical Chemistry (42):12,1930-1937. www.cellavision.com Wright D., (2010): Abbott Learning Guide, Abbott Laboratories HM_09_39649/v2-3. www.abbottdiagnostics.com/www.beckman.com/products/a pplications/partChar/ CoulterPrinciple_dcr.asp. Paterakis G. et al, (1998): Comparative Evaluation Of The Erythroblast Count Generated by Three-Color Fluorescence Flow Cytometry, The Abbott CELL-DYN 4000 Hematology Analyzer, and microscopy, Int Journal of Laboratory Hematology (4):6470.
3.
4.
5.
6. 7. 8. 9.
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10.
11. 12. 13.
Briggs C. et al, (2007): Continuing Developments with the Automated Platlet Count, Int Journal of Laboratory Hematology (2):77-91. www.nlm.nih.gov/medlineplus/ency/article/003344.htm\. Harmening D., (2002): Hemtology And Fundamentals of Hemostasis , F.A. Davis, fourth edition. Harmening D., (2002): Modern Blood Banking and Transfusion Practice, F.A. Davis, fourth edition.
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INDEX
antigen ....... 12, 84, 89, 91, 92, 94, 145, 150, 153 antigens (Ags) ................................... 91 antiglobulin ..................................... 106 anti-gram ......................................... 108 anti-human globulin ...... 103, 105, 106 aperture ...........................27, 28, 35, 66 Aqueous methylene blue .................. 29 as methylene blue ............................. 51 aspirin.......................................... 74, 76 autoantibody ................................... 113 auto-logous .................... 110, 112, 113 auto-logous control ......................... 112 Auto-logous Control ...................... 110 auto-logus ....................................... 112
A
ABO and Rh-Grouping ............89, 97 ABO antibodies .................................90 ABO compatibility .......................90, 91 ABO grouping ..................................90 ABO groupings .................................89 Acetic Acid. Glacial .........................29 Activated Partial Thromboplastin time (aPTT .................................79 agglutination ... 84, 90, 93, 94, 98, 101, 102, 103, 104, 106, 111, 112, 113, 122 Agreen metallic sheen ......................51 AHG .....103, 104, 106, 111, 112, 113, 114 AHG reagent ...................................113 allergic conditions ............................41 alloantibody ....................................112 Ammonium oxalate ..........................41 Ammonium Oxalate ............ 17, 18, 41 ammonium salt with sodium chloride ......................................................61 anaemia .......................................46, 57 antecubital fossa. ..............................74 antibodies ..... 12, 28, 43, 89, 90, 91, 94 anticoagulation ..................................15 anti-D reagent ...................................97
B
Basophils .................................. 12, 128 bleeding time ......... 41, 74, 75, 76, 148 bleeding time (BT) ........................... 74 Blood Cell Counting..................... 7, 19 Blood Film .................................... 7, 49 bone marrow .. 9, 13, 44, 125, 151, 154 Brilliant cresyl blue .......................... 45 buffer .....................................36, 51, 55
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C
CaCI ............................................79, 80 Capillary blood ...........................14, 15 capital fossa veins .............................16 carbon dioxide ..................... 11, 19, 57 Carboxyhaemoglobin .......................57 CBC.............................................9, 138 compelet blood count ................... 9 CHCMr .............................................48 CHDWr .............................................48 Check Cell.......................................111 CHr ....................................................48 Chromogenic principle ....................83 chronometer ......................................83 cirrhosis .............................................41 clotting ..... 17, 18, 19, 41, 77, 148, 154 common pathway..............................79 complete blood count ......................... 9 Coombs Cells ...............................113 Coulter Principle ..... See Direct current Counting Chamber............................20 CPDA (CPDA-1) ..............................93 cresyl blue solution ...........................45 cuvette .........................................62, 63 Cyanmethemoglobin.........................57 Cynox ................................................62
DAT ... See Direct anti-human globulin Testing Panel DC ........................... See direct current Digital morphology ......................... 53 dipotassium salt ................................ 45 Direct current .............................. 26, 28 Drabkins Reagent ...................... 58, 61
E
EDTA ................ 17, 18, 45, 58, 69, 93 Eosine................................................ 51 Eosinophils .............................. 12, 129 EPO ................................................... 48 Erythrocyte Sedimentation Rate .. 8, 67 Erythrocytes ......................... See RBC ESR .............................8, 18, 67, 69, 71 Ethyl Alcohol.................................... 16 Ethylenediamine Tetra-Acetic ....... See EDTA extrinsic clotting pathway ................ 77 extrinsic pathway ........................ 77, 78
F
factors II ............... 77, 78, 81, 147, 148 factors IX .......................................... 81 factors V............................................ 81 factors VIII ....................................... 81 factors X............................................ 81 factors XI .......................................... 81 factors XII ......................................... 81 ferricyanide ....................................... 57
D
D (Rho) antigen ................................92 damaged cells....................................14
Hematology Handbook Reference & Automation Methods- First Edition
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fibrin .. 13, 77, 78, 81, 83, 95, 147, 153, 154 fibrinogen ....... 77, 79, 81, 87, 122, 154 Fibrinogen .........................................95 filter paper ...................................74, 75 FIVE PART DIFF ..........................37 five-part differential....................35, 36 Flow cytometry .................................33 fluorochrome propidium iodide .......49 formaldehyde ..............................20, 36
HMWK ............................................. 81
I
IDAT ............................................... 103 idiopathic thrombocytopenic purpura (ITP) ............................................. 41 IgG ................................. 105, 112, 114 IgM .................................................... 90 Immature granulocyte (IG) ............ See Immature granulocyte (IG) Immature granulocyte (IG)" ......... 37 immature Reticulocyte Fraction ....... 48 immunological humeral response .... 12 Impedance counting ......................... 26 in vitro ............................................... 17 in vivo.......................................... 17, 90 infection 12, 15, 28, 37, 130, 136, 148, 149 Internal structure............................... 33 IYT .................................................. 103
G
Giemsa stain............... 54, 55, 116, 118 Glanzmanns thrombasthenia ...........76
H
Haematocrit ...................... 8, 18, 64, 65 haemoconcentration ..........................16 Haemocytometer .........................20, 23 haemoglobin 19, 48, 57, 62, 63, 64, 72 Haemoglobin content of reticulocytes (CHr) ............................................48 Haemoglobinometer .....................8, 62 haemoglobin-S ..................................72 haemolyses ........................................15 HDWr ................................................48 Hematology....... 1, 5, 6, 8, 9, 138, 139, 147, 149, 151, 153 Hematopoiesis .................................13 hemolysis 71, 87, 90, 98, 101, 148, 154 Heparin ........................................17, 19
K
kallikrein ........................................... 81 Keeping Time of EDTA Blood ........ 18
L
Landsteiner's rule .............................. 89 Latex microparticles ......................... 84 Leishmania.............................. 7, 55, 56 leucocytosis ...................................... 29
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Leukocytes ........................... See WBC lungs ..................................... 11, 19, 57 lymph ................................. 9, 150, 151 Lymphocytes ...................................12 lyse-resistant RBC ............................33
O
oxygen ............ 11, 19, 57, 72, 148, 149
P
Packed Cell Volume, PCV ............. 64 para-nitroaniline (pNA) .................... 83 peroxidase ......................................... 36 phagocytosis ..................................... 28 Phosphate Buffer .............................. 51 photoelectric colorimeter ................. 58 Photo-optical principle .................... 82 plasma 9, 13, 15, 65, 68, 69, 77, 78, 79, 80, 81, 82, 89, 91, 94, 122, 131, 146, 147, 154 platelet ...... 41, 42, 43, 53, 74, 76, 134, 135, 149, 154 Platelet Counts ................................ 41 PLT-o = RNA content ...................... 42 pneumonia .................................. 38, 41 polymethine ...................................... 48 positive Ab screen .......................... 112 positive agglutination ..................... 111 positive antibody screen ................. 107 Potassium Cyanide ........................... 58 Potassium Ferricyanide .................... 58 Potassium Oxalate ...................... 17, 18 Prothrombin time .................... 8, 77, 79 puncture ................................14, 16, 58
M
Malaria ....................................7, 54, 56 MCH ...........................................19, 26 MCHC ........................................19, 26 MCV .......................... 19, 25, 116, 118 MCVr ................................................48 Methaemoglobin ...............................57 methyl alcohol ..................................51 Monoclonal antibodies .....................43 Monocytes ................................12, 130 Multiple Abs ...................................113 Myeloperoxidase ..............................36
N
nephelometric principle ....................82 Nephelometric principle ..................82 neubauer counting chamber .............41 Neubauer counting chamber ............20 Neutrophils ..............................12, 132 Nucleated Red Cells ........................31 Nucleic acid content (RNA and DNA) ......................................................33 Nucleic acid dye Oxaz ......................48
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R
RDWr ................................................48 RETHE............................................48 Reticulocyte ............. 7, 15, 44, 48, 118 Reticulocyte haemoglobin ................48 Reticulocyte Volume (MRV) ...........48 rouleau formation .............................71 rouleaux. Formation .........................94
S
screening cells ........ 107, 108, 110, 111 serum . 15, 89, 91, 92, 93, 97, 102, 145, 153 Sickle cell anaemia .....................46, 72 Sickle Cell Test .................................72 Simplate ............................................74 single Ab .........................................113 Sodium Bicarbonate .........................58 sodium laurryl sulfate .......................61 sodium metabisulfite ..................72, 73 spectrophotometer ......................58, 59 Sphygmomanorneter .........................74 spleen ................... 9, 14, 121, 153, 154 Spleen ...............................................14 splenectomy ............. 41, 121, 149, 153 Surgicutt ............................................74
the intrinsic ....................................... 79 the nodular edge of the sore ............. 55 thick film..................................... 50, 54 thrombin. ..................................... 19, 77 thrombocytes ....................See platelets thrombocytopenia ... 76, 134, 135, 148, 154 thromboplastin ................77, 78, 79, 80 tissue juice ........................................ 55 tourniquet .......................................... 16 Trisodium citrate ..................18, 20, 45 Trisodium Citrate .................17, 18, 69
V
VCS .....................................35, 47, 138 venous blood .................15, 45, 65, 150 Venous Blood ............................... 7, 15 venous method .................................. 16 vitamin K .................................... 78, 82
W
Washed Cell Suspensions ............... 94 Wharton's jelly .................................. 94 Wrights stain ......................... 7, 50, 51
X
X100 objectives ................................ 52 X40 objectives .................................. 52
T
thalassaemia ......................................31
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GLOSSARY OF TERMS
anemia: Low red blood cell (RBC) count or hemoglobin in blood; caused by blood loss or by impaired production or destruction of RBCs antibody: A protein molecule in serum or body fluids that reacts with, protects against, and helps destroy foreign or natural substances (antigens) antigen: Any substance, either foreign or natural, that stimulates lymphocytes (white blood cells) to initiate an immune response; includes bacteria, viruses, fungi, toxins, living and dead tissue, etc. aplastic: Characterized by incomplete or defective development basophil: A cell that stains specifically with basic dyes -blast: A suffix indicating an immature cell calibrator: A known quantity of an analyte used to establish a normal curve catalyst: A substance whose presence changes the velocity of a reaction but does not form part of the final product of the reaction; the verb form is catalyze chemistry: In clinical testing, refers to the solutes dissolved in the plasma such as uric acid, etc. coefficient of variation (CV): A statistical term that indicates the precision or reproducibility of a measurement; expressed as percentages, CVs indicate the degree of small variations between the
145
same tests run on the same sample (the smaller the number, the more precise the instrument or test) congestive heart failure (CHF): A collection of symptoms indicating that the heart is unable to pump effectively; inadequate blood circulation results in decreased oxygenation of tissues and breathlessness control: In chemistry, a known quantity of an analyte that is tested as if it were an unknown to find out how well the instrument is performing correlation coefficient (r): A value indicating accuracy; values closest to 1.00 are best -cyte or cyto-: Combining forms meaning cell dialysis (also called hemodialysis): The process of removing toxins and excess water from the blood; used in kidney failure diapedesis: The process by which cells squeeze through the pores of a membrane diffusion: The movement of particles from an area of greater concentration to an area of lesser concentration ecchymosis: Bruising electrolyte: Any substance that, in solution, becomes an ion and conducts electricity; examples are sodium and potassium -emia: Suffix that can be interpreted as "in the blood"
146
end point: Term for a reaction that is measured at the end; for example, a test converts all of a substance present in a specimen into a chromogen which has a specific color and then measures the color enzymes: Proteins that catalyze chemical reactions; many enzymes are present in one organ; enzymes appear in blood because they have a natural function there or because disease in the tissue in which they originate caused them to be dispersed via the blood epistaxis: Nosebleed erythro-: Prefix meaning red erythroblast: Literally, an immature red blood cell erythropoiesis: Red blood cell formation ferritin: The primary form of storage iron fibrin: A plasma protein that acts with other blood factors to create blood clots glucose: The sugar that is the energy source for all body cells granulopoiesis: The formation of granulocytes hema- or hemo-: Prefixes indicating blood hematology: Literally, the study of blood; refers to the gross features of blood such as cell counts, bleeding time, etc.
147
hematopoiesis: Formation of blood cells hemoglobin: The oxygen-carrying component of red blood cells; composed of two pairs of protein chains called globin and four smaller units called heme, which contain iron hemolysis: Destruction of red blood cells, often by separation of hemoglobin; caused by many substances or by freezing or heating hemolytic-uremic syndrome (HUS): A sudden disorder that involves thrombocytopenia, hemolysis, and acute renal failure; HUS primarily occurs in infants and children following an infection but occasionally occurs in adults; most patients recover, but some require renal dialysis hemophilia: A bleeding disorder due to hereditary deficiencies in the blood clotting factors hemostasis: The process of stopping bleeding hepat-: Prefix indicating the liver hepatitis: An inflammation of the liver histiocytes: Macrophages in the connective tissue; part of the reticuloendothelial system histogram: A graphic means of comparing magnitudes of frequencies or numbers of items; usually shown in bar graphs or columns homeostasis: The body's tendency to maintain a uniform or stable state
148
hormones: Endocrine gland secretions that travel to and act on specific target organs hyper-: Prefix indicating abnormally increased values or activity hypo-: Prefix indicating abnormally decreased values or activity hypochromia: A decrease of hemoglobin in the red blood cells so that they are abnormally pale hypoxia: Abnormally low oxygen level idiopathic: Occurring without known cause idiopathic thrombocytopenic purpura (ITP): A chronic blood disorder with no apparent cause (except in children when it may follow a viral infection); the body develops an antibody directed against platelet antigens, causing bleeding (petechiae, purpura, or mucosal bleeding) that may be minimal or extensive; treatment options include steroids, infusion of platelet concentrates, or splenectomy interstitial fluid: Fluid surrounding cells; transports nutrients, gases, and wastes between blood and cells ion: An atom or molecule that carries either a positive (cation) or negative (anion) electrical charge -itis: Suffix indicating inflammation lymphoid: Associated with lymph; refers to the lymphatic system and to the fluid collected from tissues that flows through the lymph vessels and is added to venous blood
149
lyse: To break up to cause cells to disintegrate lysin: An antibody that acts destructively on cells depending on the antigen that stimulated its production lysis: The destruction of red blood cells, bacteria, and other antigens by a specific lysin macro-: Prefix meaning large; for example, a macrocyte is a large cell megalo-: Prefix meaning large; for example, a megalocyte is a large cell menorrhagia: Excessive menstrual bleeding meta-: Prefix indicating after or behind; this prefix has the same meaning as the prefix postmetabolism: Physical and chemical processes by which a living organism breaks down complex substances into simpler substances for nutritional use or disposal microcyte: A red blood cell that is five microns or less in diameter microcytic, hypochromic: Adjectives describing a form of anemia with red blood cells that are small and pale mye-: Prefix meaning bone marrow myeloid: Associated with the bone marrow neoplasm: Any new or abnormal growth
151
nephr-: Prefix indicating the kidney nephritis: Inflammation of the kidney normo-: Prefix meaning normal or usual normochromia: Indicating red blood cells having normal coloring normocyte: A red blood cell that is normal in size, shape, and color normocytic, normochromic: Adjectives describing normal cells with normal coloring; used to indicate the status of red blood cells -oma: Suffix indicating a tumor or neoplasm; for example, lymphoma means tumors affecting the lymph system -osis: Suffix indicating a condition; for example, thrombocytosis is a condition affecting thrombocytes (platelets) pancytopenia: A condition in which the numbers of all types of blood cells are reduced panel tests: Usually, a group of three to five tests involving one organ system (heart, liver, kidneys, etc.) or having reference to one condition; used to confirm a diagnosis, to monitor a condition or disease state, or to modify therapy -penia: Suffix indicating a decreased amount petechiae: Pinpoint bleeding into the skin from broken blood vessels usually on arms or thighs without trauma
151
pH: means of indicating the acidity of body fluids; pH is measured on a scale of 0 (highly acidic) to 14 (highly alkaline), with 7.0 as neutral; normal range for blood pH is 7.35 to 7.45 phagocyte: A cell that ingests bacteria, foreign particles, and other cells to protect the body polycythemia: Literally, many cells in the blood; in this condition, which is the opposite of anemia, the blood becomes highly viscous (thick) and flows sluggishly polymorphonuclear: Having various forms of nuclei porphyria: A group of inherited conditions in which the production of heme is deficient pro-: Prefix indicating before or a precursor profile: In chemistry, a group of tests that can be used to screen for an abnormality that may not be readily detected in another way; often involves 12 to 28 tests performed on a venipuncture sample purpura: Hemorrhagic disease characterized by the escape of blood into the tissues, under the skin, and through the mucous membranes; results in spontaneous bruises and small red patches on the skin rate reaction: Term referring to a measure of the rate of activity caused by the presence of an analyte (the constituent or substance that is analyzed); the more analyte in the specimen, the more activity occurs in a specific period of time
152
reference method: Method of comparing accuracy between two or more testing systems or kits reference range: Normal values; ranges of values for each assay for people in a defined population renal: Pertaining to the kidneys reticul-: Prefix indicating a network reticulocyte: A young red blood cell containing a network of basophilic substances rheumatoid arthritis: An incurable inflammatory disease of the joints and connective tissue; an autoimmune disease serology: The study of antigen-antibody reactions in blood samples (as opposed to within the body) serum: The fluid portion of the blood after fibrin and the formed elements have been removed splenectomy: Surgical removal of the spleen systemic: Pertaining to or affecting the body as a whole systemic lupus erythematosus (SLE): An incurable inflammatory disease affecting many body systems; an autoimmune disease thrombin: An enzyme that converts fibrinogen to fibrin thrombo-: Prefix indicating blood clot
153
thrombocytopenia: Reduced platelet count thrombocytosis: Elevated platelet count thrombotic thrombocytopenic purpura (TTP): An acute and potentially fatal disorder, TTP involves fragmented RBCs, severe thrombocytopenia, hemolysis, elevated reticulocyte count, fever, and possible damage to many organs caused by lack of adequate blood flow. Cause is unknown; without therapy (repeated administration of large volumes of plasma), TTP is usually fatal timed reaction: Term for test measurements such as blood coagulation tests in which clotting times are calculated from reaction rates transferrin: A blood protein synthesized by the liver; transferrin binds to iron, transporting and releasing it to storage tissues (liver, bone marrow, and spleen) uremia: An excess of nitrogen waste in the blood venipuncture: Blood taken from a vein
154
Note:
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Hematology Handbook Reference & Automation Methods

Hashem A. Abu-Hurirah, BSc (MT), MSc, PhD fellow

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hemoglobin Determination... 7.1. Cyanmethemoglobin Method. 7.2. Hemoglobinometer.

Hematocrit. Erythrocyte Sedimentation Rate (ESR)..

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Keeping Time 24 hr 24 hr 1 hr 1 hr 2 hr 24 hr 1 hr 3 hr 1 hr 48 hr 1hr

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Figure 4.2: Improved Neubauer haemocytometer counting chamber ruling.

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Red Cell Indices:

Normal range: 76-96 fl MCV = PCV x 10 RBC Count in Million

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Figure 4.5: Explanation of Direct current small opening (aperture).

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Figure 4.6: Explanation of optical system (Laser light scatter)

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Eosinophilic Segmented Cell

Basophilic Segmented Cell

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

4.4 Reticulocytes Count

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Reference values Adults & children Infants 0.2 - 2.0% 2 - 6%

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

C. Malaria plasmodium falciparum

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE

No. of pulses = count of particle of particle Average pulse Height = volume

Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE