T H E E F F E C T S O F I N V I T R O H E M O L Y S I S O N T H E C O M P R E H E N S I V E M E T A B O L I C P A N E L A Report of Senior Thesis by Brent Thomas Carl Robinson Major: Biology Maryville College Fall, 2002 Date Approved , by Faculty Supervisor , by Editor ii ABSTRACT Hemolysis is a common problem in clinical chemistry analyses because it interferes in the determination of several chemical parameters. Using fully mechanized autoanalyzers, the effects of hemolysis in the determination the 16 clinical blood chemistry parameters that are included in the comprehensive metabolic panel were determined quantitatively by adding hemolysate to serum. Free hemoglobin concentrations up to 800 mg/dL did not significantly affect the following analytes: chloride ion, sodium ion, calcium ion, blood urea nitrogen, creatinine, alkaline phosphatase, conjugated bilirubin, and carbon dioxide. However, hemolysis at 800 mg/dL of free hemoglobin and higher caused erroneously high values for: potassium ion, glucose, total bilirubin, unconjugated bilirubin, total protein, and albumin. Hemolysis at 400 mg/dL of free hemoglobin and higher caused significantly high values in aspartate aminotransferase, and significantly low values in alanine aminotransferase. These results differ subtly from those of other studies; therefore, it is extremely important for each laboratory to know how hemolysis affects its chemical analyses because iii the effects vary among methodologies. After such determination, hemolysis should be accounted for when interpreting results and in diagnosis. iv TABLE OF CONTENTS Page Chapter I Introduction ............................... 1 Components of the Blood ............... 1 Clinical Chemistry .................... 16 Hemolysis ............................. 23 Purpose and Hypothesis ................ 27 II Materials and Methods ...................... 28 III Results .................................... 32 IV Discussion ................................. 38 Potassium ............................. 40 Glucose ............................... 41 Aspartate Aminotransferase ............ 42 Total, Unconjugated, and Conjugated Bilirubin ............................. 43 Total Protein ......................... 44 Albumin ............................... 44 Alanine Aminotransferase .............. 45 Urea Nitrogen, Calcium, Sodium, Creatinine, Carbon Dioxide, Alkaline Phosphatase, and Chloride ............. 46 MLT Qualitative Grades ................ 47 Conclusions ........................... 50 Appendix .. ....................................... 53 References ....................................... 63 v LIST OF TABLES Table Page 1 Functions of Formed Elements of the Blood............................ 2 2 Normal Values of the Individual Determinations within the Comprehensive Metabolic Panel in Adults............................... 18 3 The Comprehensive Metabolic Panel Analyses and Methodology in the Ortho-Diagnostics Vitros 950............ 31 4 Intracellular Hemoglobin Concentrations of the 5 Participants............................ 32 5 MLT Visual Hemolysis Grades vs. Actual Grades....................... 33 6 Mean Results (± 1 SE) of the Comprehensive Metabolic Panels for Each Degree of Hemolysis ........... 35 vi ACKNOWLEDGEMENTS I would like to thank the following employees of the Blount Memorial Hospital Laboratory who helped in performing the analyses: Mary Corbitt, George Easton, Karen Easton, and James Houser. I would also like to thank the following employees of the Blount Memorial Hospital Laboratory who were participants in the study: Nathena Bruce, Karen Easton, Kim Bailey, Donna Sweitzer, and Kris Loggins. Thank you to John Bleazey, Blount Memorial Hospital Laboratory Manager, for authorizing the conductance and financing of the study. In addition, I would like to thank Dr. Drew Crain for his time and effort in supervising my study and for providing a very structured agenda that was helpful in completing the study. Finally, I would like to thank my wife Michelle and my son Brock for tolerating my diverted attention from them during the course of this study. vii 8 9 CHAPTER I INTRODUCTION Components of Blood Blood is composed of plasma and formed elements. Plasma is the protein-rich, acellular fluid in the blood. The formed elements are erythrocytes, also called red-blood cells, leukocytes, also termed white- blood cells, and platelets. Functions of the formed elements are summarized in Table 1. Around 55% of blood is plasma, and about 45% is red blood cells; platelets and white blood cells make up only about 1% of the blood (Vick, 1984). When blood is centrifuged, the more dense formed elements go to the bottom and the less dense plasma sits on the top. The percentage of formed elements in the blood is called the hematocrit (Fox, 1999). On average adult humans have just over ten pints of blood. The bright red blood flowing from the heart to the capillaries via the arteries is called arterial blood. It is oxygen enriched which yields the bright red color. After the blood has been oxygen depleted 1 Table 1. Functions of Formed Elements of the Blood. Component Function Erythrocyte Transports oxygen and carbon dioxide Leukocytes Granulocytes 1. Neutrophil Phagocytic 2. Eosinophil Helps detoxify foreign substances; secretes enzymes that dissolve clots; fights parasitic infections 3. Basophil Releases anticoagulant heparin Agranulocytes 1. Monocyte Phagocytic 2. Lymphocyte Provides specific immune response, including antibodies Platelet Enables clotting, releases serotonin causing vasoconstriction Source: (Fox, 1999, p. 368). in the capillaries, it is returned to the heart by the veins. This darker blood is termed venous blood. 2 Erythrocytes Erythrocytes are shaped like flattened biconcave discs (Fox, 1999). This unusual shape increases substrate surface area and aids in oxygen binding, which allows oxygen transport throughout the body. The main function of the erythrocytes is to circulate oxygen, which is bound to hemoglobin in the lungs and transported to the rest of the cells in the body. Another function of hemoglobin is that it is an excellent acid-base buffer, which makes the erythrocytes accountable for roughly 70% of the buffering capacity of the blood. Additionally, red blood cells contain a large amount of carbonic anhydrase, which acts as a catalyst in the reaction of carbon dioxide and water (Guyton & Hall, 2000). The catalysis of this reaction allows blood to react with vast amounts of carbon dioxide and, in turn, transport it back to the lungs. Red blood cells, although having a biconcave disk-like shape, are very amoeboid in nature. They can change shape dramatically when flowing through the capillaries (Guyton & Hall, 2000). The reason the shape of the erythrocytes is so adaptable is that the cell membrane is very excessive compared to the material inside the cell, so the cell is not filled to capacity. This allows the erythrocyte to be plastic. 3 Mature erythrocytes are also anucleated and lack an endoplasmic reticulum. Erythrocyte production In the early fetal months, red blood cells are produced in the yolk sac (Vick, 1984). As the yolk sac recedes, the liver, spleen, and lymph nodes assume the responsibility of erythrocyte production. By the fifth month, the bone marrow begins to produce red cells, and by the time of birth, the bone marrow exclusively produces the erythrocytes (Guyton & Hall, 2000). Early in life all of the bones produce red blood cells, but as they are not needed, the longer bones become infiltrated with fat. By age twenty, only the bones of the axial skeleton produce erythrocytes. The bone marrow contains hematopoetic, or blood-forming, tissue. These tissues are separated by venous sinuses that drain into a central vein. In addition, arterial blood flows through the tissue and drains into the central vein via the venous sinuses (Vick). The bone marrow contains primordial stem cells that can lead to the formation of many types of cells. These are responsible for producing the hemocytoblasts. The genesis of red blood cells begins with the hemocytoblast. The hemocytoblast forms the basophil erythroblast which begins to produce hemoglobin (Guyton & Hall, 2000). The basophil 4 erythroblast gives rise to the polychromatophil erythroblast, and as the nucleus becomes smaller while even more hemoglobin is being produced, the cell becomes a normoblast. After the concentration of hemoglobin in the cell reaches its maximum, the normoblast then completely loses its nucleus, while at the same time the endoplasmic reticulum is shrinking. At this point the cell is an immature red blood cell, or a reticulocyte. The reticulocytes make their way into the capillaries by moving through the pores in the membrane. After the reticulocyte passes into the bloodstream, it continues to make hemoglobin for a couple of more days until the endoplasmic reticulum is completely reabsorbed (Guyton & Hall). At this point, the cell is now a mature red blood cell, or erythrocyte. Once mature, the erythrocytes circulate approximately 120 days before destruction, and red cells are synthesized at a rate higher than any other tissue in the body (Vick, 1984). There are two essential nutrients that aid in erythrocyte genesis: vitamin B12 and folic acid. Both of these vitamins are needed for DNA synthesis; thus, deficiency in these vitamins causes failure of nuclear maturation and division. Without this ability, red cells cannot be produced efficiently. As a result of this deficiency, erythrocytes become macrocytes, which are large,