Osmotic Resistance & Plasma Proteins PDF

PA GE PAGE 1 1 Osmotic resistance. Plasma https://edu.umch.d e www.umfst...

PA GE PAGE 1 1 Osmotic resistance. Plasma https://edu.umch.d e www.umfst.ro proteins. Lecturer Dr. Florina Gliga 2024 Assist. Dr. Horațiu Sabău Assist. Dr. Andreea Tinca Osmotic resistance- PAGE 2 Introduction Changes in cell volume can lead to cell damage and death. Cells have developed mechanisms to regulate their volume. Most cells are highly permeable by water because of the presence of aquaporins in their plasma membranes. Osmotic pressure gradients across the cell membrane that are generated by effective osmoles cause water to move either into or out of the cell, which result in changes in cell Medical Sciences, Naish, Jeannette. Pages 1-14. © 2019. volume.  Osmotic PAGE 3 resistance Osmotic fragility (or osmotic resistance) of red blood cells represents their sensitivity to changes in osmotic pressure. In red blood cells suspended in an isosmotic solution (0.9% NaCl solution) no visible modifications occur. Exposed to a hyposmotic solution, red cells take in increasing quantities of water, swell until the capacity of the cell membrane is exceeded and burst releasing hemoglobin. Exposed to a hyperosmotic solution, red cells give up intracellular fluid and shrink. Osmotic fragility tests are based on the measure of red blood cell lysis as a function of osmotic stress (hyposmotic solution). Osmotic PAGE 4 resistance Effects of: isotonic (A) hypertonic (B) hypotonic (C) solutions on cell volume. Guyton and Hall Textbook of Medical Physiology, Hall, John E., PhD; Hall, Michael E., MD, MS. © 2021. Osmotic PAGE 5 resistance Cell Biology, Pollard, Thomas D., MD; Earnshaw, William C., PhD, FRS. Pages 227-239. © 2017. Osmotic resistance PAGE 6 Medical Physiology, Aronson, Peter S.; Boron, Walter F.; Boulpaep, Emile L. © 2017. Osmotic resistance PAGE 7 Medical Physiology, Aronson, Peter S.; Boron, Walter F.; Boulpaep, Emile L. © 2017. Osmotic resistance PAGE 8 1. Normal values: – minimum osmotic resistance: 0.44% (4.4 g/l), – maximum osmotic resistance: 0.3-0.32% (3-3.2 g/l) 2. Higher values: increased osmotic fragility / decreased osmotic resistance: – decreased surface area to volume ratio of erythrocytes (spherocytosis, elliptocytosis) –altered red blood cell membrane (aging red blood cells due to decreased turnover, autoimmune hemolytic anemia). 3. Lower values: decreased osmotic fragility / increased osmotic resistance: – increased surface area to volume ratio of the red cells (thalassemia, iron deficiency). Plasma proteins PAGE 9 Total plasma protein In very general terms, variations in plasma protein concentrations can be caused by changes in any of three factors: 1. the rate of protein synthesis 1. the rate of removal 1. the volume of distribution. Plasma proteins PAGE 10 The role of proteins To maintain a normal distribution of water between the vascular and extravascular (interstitial) spaces. This is determined by the equilibrium between hydrostatic pressure, which tends to force fluid out of the capillaries, and oncotic pressure, which acts to retain fluid within the vessel. The net flow of fluid outwards is balanced by ‘suction’ of fluid into the lymphatics, which returns it to the bloodstream. Similar principles govern the volume of the peritoneal and pleural spaces. Plasma proteins PAGE 11 The role of proteins Robbins Basic Pathology. Kumar, Vinay, MBBS, MD, FRCPath; Abbas, Abul K., Textbook of Histology, Gartner, Leslie P., PhD. Pages 65- MBBS. Pages 97-119. © 2018. 78.e3. © 2021. Plasma proteins PAGE 12 Plasma proteins can be classified into two groups: 1. albumin 2. globulins. Can use total protein and albumin to calculate an A/G ratio: Globulin= total protein (g/dl)- albumin (g/dl) A/G Ratio= albumin (g/dl)/ globuline (g/dl) Normal A/G= 1.0-1.8 High A/G suggests underproduction of globulins found in some leukemias and genetic deficiencies. Low A/G - suggests overproduction of globulins ( multiple myeloma or autoimmune disease) or underproduction of albumin (liver disease or nephrotic syndrome). Plasma proteins PAGE 13 Methods to measure Total proteins and Albumin Medical Laboratory Science Examination Review, by Linda Graeter, PhD, Elizabeth Hertenstein, Charity Accurso and Gideon Labiner, Elsevier  PAGE 14 Total proteins- Biuret method Method of determination The principle of this method is that the peptide bond of amino acid residues and two copper ions (Cu2+) can form a purple complex in alkaline condition, and the darkness of the color is proportional to the content of protein in the sample. The absorbance of the solution is normally measured at 546 nm base on spectrophotometric method.  PAGE 15 Total proteins- Biuret method Steps Zheng, Kangle, et al. "Measurement of the total protein in serum by biuret method with uncertainty evaluation." Measurement 112 (2017): 16-21. Serum protein electrophoresis PAGE 16 Indications the quantitation of specific proteins the relative increases and decreases within the protein population (in the diagnosis of immunodeficiency diseases and immunoproliferative disorders) information about the homogeneity of a fraction Protein electrophoresis can be performed from: 1. serum from whole blood 2. urine 3. cerebrospinal fluid Serum protein electrophoresis PAGE 17 Protein electrophoresis is a standard laboratory technique by which charged protein molecules are transported through a solvent by an electrical field. Electrophoresis can be used for the separation of a wide variety of charged molecules: amino acids polypeptides proteins DNA. Most biological molecules carry a net charge will migrate at a rate proportional to their charge density. A guide to polyacrylamide gel electrophoresis and detection- BIO-RAD.  PAGE 18 Serum protein electrophoresis The mobility of a molecule through an electric field will depend on the following factors: the electrical field strength the net charge on the molecule the size and shape of the molecule the ionic strength the properties of the matrix through which the molecule migrates (viscosity, pore size). Proteins come in a wide range of sizes and shapes and have changes imparted to them by the dissociation constants of their constituent amino acids.  PAGE 19 Serum protein electrophoresis Protein separation by electrophoresis: A porous support, such as paper, cellulose acetate, or a polymeric gel, is commonly used to minimize diffusion and convection. On a wet cellulose acetate foil, the proteins are separated according to their net charge. If an alkaline pH is used, as in this case, the proteins are negatively charged and move to the anode. Principles of Medical Biochemistry, Meisenberg, Gerhard, PhD; Simmons, William H., PhDPages 18- 32. © 2017. Serum protein electrophoresis PAGE 20 Steps in the standard performance of serum protein electrophoresis (SPE): 1.Separation of proteins: serum specimens are applied close to the cathode end of a support medium that is saturated with an alkaline buffer (pH 8.6) the support medium is connected to two electrodes and a current is passed through the medium to separate the proteins. all major serum proteins carry a net negative charge at pH 8.6 and migrate toward the positive terminal, the anode. Serum protein electrophoresis PAGE 21 1.Separation of proteins: Using SPE, serum proteins separate into five bands: albumin α1 –globulins α2 –globulins β-globulins γ-globulins. The width of the protein band in a particular fraction depends on the concentration of proteins present in that fraction; homogeneous protein gives a narrow band. Medical Biochemistry, Dominiczak, Marek H. © 2019. Serum protein electrophoresis PAGE 22 2. Fixation of proteins: The protein fractions are fixed by immersing the support medium in an acid solution (acetic acid): immobilize the proteins. 3. Protein staining: In order to visualize the bands, the proteins are then stained. 4. Reading the values: Densitometric scan - The absorbances are normally recorded on a strip-chart recorder to obtain a pattern of the fractions. Serum protein electrophoresis PAGE 23 Ferri's Clinical Advisor 2021, Castillo, Jorge J., MD; Ghobrial, Irene M., MD. Published January 1, 2021. Pages 918-920.e1. © 2021. Serum protein electrophoresis PAGE 24 A reference serum control is processed with each electrophoretic run. To assess the molecular masses (sizes) of proteins in a gel, a prepared mixture containing several proteins of known molecular masses is run alongside the test sample in one or more lanes of the gel. Such sets of known proteins are called protein molecular weight (or mass) markers or protein ladders. thermofisher Serum protein electrophoresis PAGE 25 Classes of proteins: 1.Albumin: negative acute phase protein nonspecific binder of many substances major function is to maintain colloid osmotic pressure. Medical Laboratory Science Examination Review, by Linda Graeter, PhD, Elizabeth Hertenstein, Charity Accurso and Gideon Labiner, Elsevier Serum protein electrophoresis PAGE 26 2. Globulins: Includes α1, α2, β and γ fractions. A.α1 Region: - α1-Antitrypsin: positive acute phase protein; major component of the a1 region (90%); functions to inhibit protease neutrophil elastase; deficiency is associated with a mutation - associated with emphysema and cirrhosis). Serum protein electrophoresis PAGE 27 2. Globulins: B. α2 Region - Haptoglobin: Positive acute phase protein; serves to bind free hemoglobin in the blood for clearance; used clinically to detect and evaluate hemolytic anemia - Ceruloplasmin: Positive acute phase protein, functions to bind serum copper (90% bound by ceruloplasmin), used clinically to aid in the diagnosis of Wilson’s disease - Other includes α2-macroglobulin Serum protein electrophoresis PAGE 28 3. Globulins: C. β Region - Transferrin: Negative acute phase protein; functions in iron transport to the tissues; used clinically to investigate causes of anemia, gauge iron metabolism, and determine iron-carrying capacity in the blood D. γ Region - Immunoglobulins: Includes IgG, IgA, IgM, IgD, and IgE Serum protein electrophoresis PAGE 29 Mechanism of positive and negative acute phase proteins. Medical Biochemistry Dominiczak, Marek H. © 2019. Serum protein electrophoresis PAGE 30 CRP is absent from a healthy person. CRP increased after any injury (trauma, bacterial infection, surgery, neoplasm, and inflammation) to 100 times. 1. This is a nonspecific acute-phase protein. 2. CRP rises after 4 to 6 hours of the infection (within 24 hours) 3. The peak level is at 72 hours. 4. C-Reactive Protein (CRP) becomes negative after 7 days. 5. typically migrates between β and γ region during electrophoresis. Medical Biochemistry Dominiczak, Marek H. © 2019. Serum protein electrophoresis PAGE 31 Normal electrophoretic pattern Clinical Diagnosis and Management by Laboratory Methods. Philadelphia, WB Saunders, Henry JB , 2001  Serum protein electrophoresis PAGE 32 Common abnormal electrophoretic patterns Acute phase reaction: Increased bands in the a1, a2, and beta regions with a decreased albumin band; seen with infection, tumor growth, hepatitis, surgery, trauma, burns Clinical Diagnosis and Management by Laboratory Methods. Philadelphia, WB Saunders, Henry JB , 2001 Serum protein electrophoresis PAGE 33 Common abnormal electrophoretic patterns Nephrotic syndrome pattern: Decrease in albumin and gamma bands (the patient loses serum albumin, low-molecular weight proteins and IgG in the urine), with an increase in a2-globulins Clinical Diagnosis and Management by Laboratory Methods. Philadelphia, WB Saunders, Henry JB , 2001 Serum protein electrophoresis PAGE 34 Common abnormal electrophoretic patterns M-spike and diffuse increase in the gamma-globulin band, found in polyclonal gammopathy (multiple myeloma) Clinical Diagnosis and Management by Laboratory Methods. Philadelphia, WB Saunders, Henry JB , 2001 Serum protein electrophoresis PAGE 35 ↑: Alb: dehydration α 1 : neoplastic diseases, inflammation α 2 : neoplasms, inflammation, infection, nephrotic syndrome β: hypothyroidism, biliary cirrhosis, DM Gamma: polyclonal gammopathy (multiple myeloma) ↓: Alb: malnutrition, chronic liver disease, malabsorption, nephrotic syndrome, burns, SLE α 1 : emphysema (α 1 -antitrypsin deficiency), nephrosis α 2 : hemolytic anemias (↓ haptoglobin), severe hepatocellular damage β: hypocholesterolemia, nephrosis Gamma: immunodeficiency diseases Experiment- Osmotic fragility PAGE 36 tests Principle The osmotic resistance of erythrocytes is determined by exposing erythrocytes to different concentrations of salt solutions. The hemolysis is observed after centrifugation as a light red colored supernatant. Experiment- Osmotic fragility PAGE 37 tests Materials – 12 test tubes – 0.5% salt solution (0.5 g NaCl in 100 ml distilled water, 5 g in 1 l water, 1 g NaCl in 200 ml distilled water) – distilled water – anticoagulant-treated blood Experiment- Osmotic fragility PAGE 38 tests Procedure 1. Place the test tubes in a holder and mark them from 1 to 12. 2. Put as many drops of 0.5% salt solution in the test tubes as indicated by the marks. 3. Complete with distilled water to 25 drops. 4. Mix the content of every tube (shake the tubes). Thus a series of hyposmotic concentrations of salt solution is obtained. 5. Put one drop of packed red cells in every test tube and mix (100 microliters with the automated pipette) 6. Centrifuge the samples in order to speed up the sedimentation process. (14000 rotations, 30 degree Celcius, 3 minutes) Experiment- Osmotic fragility PAGE 39 tests Procedure 7. Observe the signs of hemolysis! Observe the color of the supernatant and of the sediment. The hemoglobin released from the disintegrated red blood cells colors the supernatant in light red. 8. Note the test tube with the less hyposmotic solution showing light red supernatant! If all of the red cells have been disintegrated, the sediment contains only the membranes and stroma of the red blood cells! Note the test tube with the less hyposmotic solution showing a small quantity of yellow sediment! Experiment- Osmotic fragility PAGE 40 tests Results Calculate the concentration of the salt solution in the test tubes. Determine the saline concentration at which lysis begins. This represents the minimum osmotic resistance. Determine the saline concentration at which lysis is complete. This represents the maximum osmotic resistance. Expected results PAGE 41 Experiment by Dr. Andreea Tinca and Dr. Horațiu Sabău  Reference PAGE 46 s 42 1. FERRI’S BEST TEST: A PRACTICAL GUIDE TO CLINICAL LABORATORY MEDICINE AND DIAGNOSTIC IMAGING SECOND EDITION, Ferri F. Fred , 2010, Elsevier 2. Berne and Levy Physiology, Koeppen, Bruce M., MD, PhD; Stanton, Bruce A., PhD. © 2018. 3. Cell Biology, Pollard, Thomas D., MD; Earnshaw, William C., PhD, FRS. Pages 227-239. © 2017. 4. Guyton and Hall Textbook of Medical Physiology, Hall, John E., PhD; Hall, Michael E., MD, MS. © 2021. 5. Medical Sciences, Naish, Jeannette. Pages 1-14. © 2019. 6. Netter's Essential Physiology, Mulroney, Susan E., PhD; Myers, Adam K., PhD, Pages 12-22. © 2016. 7. Clinical Diagnosis and Management by Laboratory Methods. Philadelphia, WB Saunders, Henry JB , 2001 8. Medical Biochemistry, Dominiczak, Marek H. © 2019. 9. Robbins & Cotran Pathologic Basis of Disease, Kumar, Vinay, MBBS, MD, FRCPath; Abbas, Abul K., MBBS. Pages 71-113. © 2021. 10. Textbook of Histology, Gartner, Leslie P., PhD. Pages 65-78.e3. © 2021. 11. Clinical Chemistry, Marshall, William J., MA PhD MSc MB BS FRCP FRCPath FRCPEdin FRSB FRSC; Lapsley, Marta, MB BCh BAO MD FRCPath; Day, Andrew, MA MSc MB BS FRCPath; Shipman, Kate, MA BMBCh MRCP FRCPath. Pages 285-301. © 2021. 12. Robbins Basic Pathology. Kumar, Vinay, MBBS, MD, FRCPath; Abbas, Abul K., MBBS. Pages 97-119. © 2018. 13. Medical Laboratory Science Examination Review, by Linda Graeter, PhD, Elizabeth Hertenstein, Charity Accurso and Gideon Labiner, Elsevier 14. Medical Biochemistry, Nagai, Ryoji; Taniguchi, Naoyuki. © 2019. 15. Ferri's Clinical Advisor 2021, Castillo, Jorge J., MD; Ghobrial, Irene M., MD. Published January 1, 2021. Pages 918-920.e1. © 2021. 16. Principles of Medical Biochemistry, Meisenberg, Gerhard, PhD; Simmons, William H., PhDPages 18-32. © 2017. 17. A guide to polyacrylamide gel electrophoresis and detection- BIO-RAD. 18. Zheng, Kangle, et al. "Measurement of the total protein in serum by biuret method with uncertainty evaluation." Measurement 112 (2017): 16-21.