CCCS4 SPEP Respiratory HE A1ATD PDF

Clinical chemistry case study Serum protein electrophoresis Respiratory system History and Examination A case study on “a1-antitrypsin deficiency” Introduction Plasma makes up 46-63% of blood Straw colored or clear Similar to interstitial fluid Differences: le...

Clinical chemistry case study Serum protein electrophoresis Respiratory system History and Examination A case study on “a1-antitrypsin deficiency” Introduction Plasma makes up 46-63% of blood Straw colored or clear Similar to interstitial fluid Differences: levels of respiratory gases Concentration and types of dissolved proteins Composition of Plasma: 92% water Proteins: for every 100ml for about 7.6 grams Albumins (60%), Globulins (35%), Fibrinogen (4%), and other plasma proteins (1%) Albumin This water-soluble protein is the most abundant of all the plasma proteins. Produced by the liver Maintains osmotic pressure of plasma Globulins 4 different kinds of globulins present in blood: alpha 1, alpha 2, beta and gamma globulin. Serve different functions including: transport substrates for forming other substances Gamma globulin: the largest portion of globulin, majority of gamma are Fibrinogen Plasma protein that functions in blood clotting Synthesized in the liver Proactive protein and is converted to fibrin in certain conditions should be absent from serum protein electrophoresis Other Plasma Proteins Remaining one 1% of plasma Peptide hormone: examples: Insulin and Prolactin Glycoproteins: examples: TSH (thyroid- simulating hormone) FSH (follicle stimulating hormone) Plasma Proteins Come From… Liver Synthesizes 90% of the proteins Lymphocytes (lymphatic system) Makes the plasma cells  antibodies Endocrine organs Peptide hormones Serum Protein ElectroPhoresis (SPEP) Serum Protein Electrophoresis (SPEP) is lab technique performed to identify types of proteins present in the blood. Venous blood sample processed into serum proteins separated by size and charge (separated into 4 types seen from last slide) This technique is useful way to diagnose some diseases. Serum protein electrophoresis on agarose gel Principle: Serum proteins are negative charged at pH 8.6 (a buffer helps to maintain a constant pH) and they move toward the anode at the rate dependent on their net charge. The speed of running depends on the amount of net charge and molecular weight of the protein molecule The separated proteins are fixed and stained by an amino acid staining dye SPEP is a type of horizontal gel electrophoresis Serum protein electrophoresis  Serum proteins electrophoresis in diagnostics of diseases Densitometry and Normal pattern A Densitometer is used for scanning of separated proteins in the gel. Scanning the pattern gives a quantitative information about protein fractions. (Scans are quantified using image processing software which can count the number of colored Major contents of each band Albumin Zone Albumin is the major fraction in a normal SPEP. A fall of 30% is necessary before the decrease shows on electrophoresis. The fastest migration towards the anode Usually a single band is seen. A decreased level of albumin, is common in many diseases, including liver disease, malnutrition, malabsorption, protein- losing nephropathy and enteropathy. Albumin - alpha-1 interzone High levels of Alpha fetoprotein (AFP) that may occur in hepatocellular carcinoma may result in a sharp band between the albumin and the alpha-1 zone. Alpha-1 zone Alpha 1 antitrypsin (A1AT) constitutes most of the alpha-1 band. Isolated band decrease is seen in the Alpha 1 antitrypsin deficiency state. The alpha-1 fraction does not disappear in alpha 1-antitrypsin deficiency because other proteins, including alpha-lipoprotein (HDL) and orosomucoid (alpha-1-acid glycoprotein, an acute phase reactant), also migrate there. Decrease combined with albumin decrease indicates protein loss (e.g. nephrotic syndrome), liver disease, malnutrition As a positive acute phase reactant, A1AT is Decreased Alpha 1 antitrypsin Decreased Alpha 1 antitrypsin Increased Alpha 1 antitrypsin Alpha-2 zone Consists principally of a-2 macroglobulin and haptoglobin. Haptoglobin: low levels in hemolytic anemia (haptoglobin is a suicide molecule which binds with free hemoglobin released from red blood cells and these complexes are rapidly removed by phagocytes). Haptoglobin is raised as part of the acute phase response, resulting in a typical elevation in the alpha-2 zone during inflammation. Increased Haptoglobin in acute inflammation Alpha-2 zone (contd.) a-2 macroglobulin (AMG) (720 Kda) elevations are seen as a sharp front to the alpha-2 band. AMG is markedly raised (10-fold increase or greater) in association with glomerular protein loss, as in nephrotic syndrome. Due to its large size, AMG cannot pass through glomeruli, while other lower-molecular weight proteins are lost. Enhanced synthesis of AMG accounts for its absolute increase in nephrotic syndrome. (hypothesized that this is a response to low albumin) Increased a-2 macroglobulin in nephrotic syndrome Nephrotic syndrome protein loss, several bands are decreased but: a2 band is increased is due to a-2 macroglobulin Beta zone Divided into Beta 1 and Beta 2 (normally Beta 1 > Beta 2) Beta 1 subzone Transferrin and beta-lipoprotein (LDL) Increased beta-1 protein due to the increased level of free transferrin is typical of iron deficiency anemia, pregnancy, and estrogen therapy. Increased beta-1 protein due to LDL elevation occurs in hypercholesterolemia. Decreased beta-1 protein occurs in acute or chronic inflammation. Increased b1 >> b2 in Iron deficiency anemia Beta zone Beta-2 subzone: comprised of Complement protein 3 and fibrinogen Complement protein 3 (C3). Increased in the acute phase response. Decreased in autoimmune disorders as the complement system is activated and the C3 becomes bound to immune complexes and removed from serum. Fibrinogen, found in normal plasma but absent in normal serum Occasionally, blood drawn from heparinized patients does not fully clot, resulting in a visible fibrinogen band between the beta and gamma globulins (SPEP should be done on a serum sample to stop fibrinogen interference) Gamma zone The immunoglobulins (Igs) are generally the only proteins present in the normal gamma region. Immunoglobulins consist of heavy chains, (A , M, G, E, and D and light chains (kappa and lambda). A normal gamma zone should appear as a smooth 'blush,' or smear, with no asymmetry or sharp peaks. The gamma globulins may be elevated, decreased, or have an abnormal peak or peaks. Note that Igs may be also be found in other zones; IgA typically migrates in the beta-gamma zone, and in particular, pathogenenic Igs may migrate anywhere, including the alpha regions. g Immunoglobulins structure 1. Antigen binding (Fab) region 2. Fragment crystallizable region (Fc) region 3. Heavy chain (blue) with one variable (VH) domain followed by a constant domain (CH1), a hinge region, and two more constant (CH2 and CH3) domains 4. Light chain (green) with one variable (VL) and one  Summery of SPEP in different disease states Acute inflammatory response Immediate response occurs with stress or inflammation caused by infection, injury or surgical trauma normal or ↓ albumin ↑ α1 and α2 globulins Some proteins of the acute-phase response. A, albumin; CRP, C- reactive protein; HG, haptoglobin; T, transthyretin (previously called pre-albumin). The latter pair can be termed negative acute-phase proteins. From Candlish JK and Crook M. Notes on Clinical Biochemistry. Singapore: World Scientific Publishing, 1993. Chronic inflammatory response Late response is correlated with chronic infection, chronic inflammation, autoimmune diseases, and cancer normal or ↓ albumin ↑α1 or α2 globulins ↑↑ g globulins Liver damage - Cirrhosis Cirrhosis can be caused by e.g. chronic alcoholism or viral hepatitis ↓ albumin ↓ α1, α2 and β globulins Increased gamma globulins (polyclonal gammopathy) b- bridging due to Nephrotic syndrome The kidney damage illustrates the long term loss of lower molecular weight proteins (↓albumin and IgG –filtered in kidney glumerulus) and retention of higher molecular weight proteins (↑↑ α2- macroglobulin) β-globulin may be increased due to increased levels of LDL (not shown) Hypogammaglobulinemia Polyclonal Gammopathy Monoclonal gammopathy Monoclonal gammopathy (also called paraproteinemia) is the presence of excessive amounts of paraprotein or single monoclonal gamma globulin in the blood. It is usually due to an underlying immunoproliferative disorder or hematologic neoplasms, especially multiple myeloma. Multiple myeloma is caused by monoclonal proliferation of β-lymphocytal clones. These “altered” β-cells produce an abnormal immunoglobulin paraprotein. The increased protein has several deleterious effects on the body, including abnormally high blood viscosity and kidney damage. Monoclonal gammopathy Diagnosis These are characterized by the presence of any abnormal protein that is involved in the immune system, When a paraproteinemia is present in the blood, there will be a narrow band, or spike, in the serum protein electrophoresis because there will be an excess of production of one protein On SPEF, paraproteins can be found in different position: between α-2 and g-fraction. (keep this in mind as a differential for isolated increased protein peaks in other zones of SPEP) Monoclonal gammopathy Types Paraproteinemias may be categorized according to the type of monoclonal protein found in blood: Light chains only (or Bence Jones protein). This may be associated with multiple myeloma or Amyloid light-chain amyloidosis Heavy chains only (also known as "heavy chain disease"); Whole immunoglobulins. In this case, the paraprotein goes under the name of "M-protein" ("M" for monoclonal). Monoclonal Gammopathies Monoclonal gammopathy on SPEP Immunofixation electrophoresis Immunofixation electrophoresis (IFE) can be used to further identify abnormal bands, in order to determine which type of immunoglobulin is present. Principle When a soluble antigen (in this case heavy or light immunoglobulin chains) is brought in contact with the corresponding antibody, precipitation occurs. Washing the excess antibodies and other proteins that are not precipitated guarantees that only the targets are fixed to the electrophoresis gel which may be visualized by staining. Monoclonal protein detection by IFE A 72 year old male who presented with lower back pain. Quantitative immunoglobulin measurements showed a large increase in serum IgG, but decreased IgA and IgM. Bone marrow exam revealed a large increase in plasma cells that were frequently aggregated. IFE on this patient's serum showed the M protein was Immunofixation electrophoresis Results interpretation Where positive precipitation reaction occurred indicates the type of the immunoglobulin. (See previous slide) Polyclonal immunoglobulins appear as diffuse bands while a monoclonal immunoglobulin appears as a narrow band IFE technique can be also applied to detect proteins other than immunoglobulins or immunoglobulin derivatives. In this case specific antibodies against those proteins are used. (See the example of fibrinogen band identification in the next slide) The respiratory system: history Cough relatively non-specific, resulting from irritation anywhere from the pharynx to the lungs. The character of a cough may give clues as to the underlying cause: Wet (productive) cough: bronchitis Dry cough: asthma or Ace inhibitors Barking coughs: croup (viral laryngotracheobronchitis) Whooping cough: pertussis The respiratory system: history Cough The character of a cough may give clues as to the underlying cause: Chronic cough:, TB, foreign body, asthma (eg nocturnal). Dry, chronic coughing may occur following acid irritation of the lungs in oesophageal reflux, and as a side- effect of ACE inhibitors. a change in character of a chronic cough; it may signify a new problem, eg infection, malignancy The respiratory system: history Haemoptysis coughed blood: frothy, alkaline, and bright red, often in a context of known chest disease vomited blood is acidic and dark Causes include: TB, bronchiectasis, pneumonia, lung abscess, COPD, sarcoidosis, hydatid, neoplasms, pulmonary embolism, vasculitis, coagulopathies, trauma/foreign body Hoarseness Abnormal change in the voice. Causes: The respiratory system: history Wheezes (rhonchi) a whistling sound caused by air expired through narrowed airways during breathing. May be monophonic (single note, signifying a partial obstruction of one airway, eg tumour) or polyphonic (multiple notes, signifying widespread narrowing of airways of differing calibre, eg asthma, COPD). Wheeze is also heard in LVF (‘cardiac asthma’). The respiratory system: history Stridor Inspiratory sound due to partial obstruction of upper airways. Obstruction may be due to something within the lumen (eg foreign body), within the wall (eg oedema from anaphylaxis, laryngospasm, croup, acute epiglottitis), or extrinsic (eg goitre, lymphadenopathy). It’s an emergency if gas exchange is compromised. https://www.youtube.com/watch?v=gYhG IFQcCnU The respiratory system: history Chest pain: usually ‘pleuritic’ if respiratory (ie worse on inspiration). Dyspnoea Subjective sensation of shortness of breath, May be hard to separate from cardiac causes; Pulmonary causes may be due to airway and interstitial disease The respiratory system: history Some scenarios in which dyspnoea is seen: Asthma may wake patient, and cause early morning dyspnoea & wheeze. Dyspnea in LVF is associated with orthopnoea and paroxysmal nocturnal dyspnoea (PND; dyspnea waking one up). Other features include ankle oedema, lung crepitations and raised JVP. Ascites can cause breathlessness by splinting the diaphragm, restricting its movement. The respiratory system: history Some scenarios in which dyspnoea is seen: Metabolic acidosis causing respiratory compensation, eg ketoacidosis, aspirin poisoning. Dyspnoea at rest unassociated with exertion, may be psychogenic: prolonged hyperventilation causes respiratory alkalosis. This causes a fall in ionized calcium leading to apparent hypocalcaemia. Features include peripheral and perioral paraesthesiae The respiratory system: history Past history: Question about pneumonia/bronchitis; TB; atopy (asthma/eczema/hay fever); previous CXR abnormalities; lung surgery; myopathy; neurological disorders, Connective tissue disorders, eg rheumatoid, SLE. Drug history: Respiratory drugs (eg steroids, bronchodilators)? Any other drugs, especially with respiratory SE (eg ACE inhibitors, b-blockers) The respiratory system: history Family history: Atopy? Emphysema? TB? Social history: Quantify smoking in ‘pack-years’ (20 cigarettes/day for 1 year = 1 pack-year). Occupational exposure (farming, mining, asbestos) Pets at home (eg birds)? Recent travel/TB contacts? The respiratory system: examination General inspection: general state (ill/well/cachexic) Color (pale, cyanosed, flushed) Breathing pattern, Accessory muscle use any coughing Neck Trachea: Central or displaced? (towards collapse, or away from large pleural effusion/tension pneumothorax; slight deviation to right is normal). Lymphadenopathy: TB/Ca The respiratory system: examination Breathing patterns The respiratory system: examination Signs of respiratory distress Tachypnoea, Nasal flaring, Tracheal tug (pulling of thyroid cartilage towards sternal notch in inspiration), Use of accessory muscles (sternocleidomastoid, platysma, infrahyoid), Intercostal, subcostal and sternal recession Face Bluish tongue and lips (central cyanosis) Conjunctival pallor (anaemia) Hands Inspect: Tobacco staining, peripheral The respiratory system: examination Hands Asterixis fine tremor (beta- agonist use), Asterixis (flapping tremor): Ask the patient to hold their hands out and push their back.wrists A tremor indicates CO2 retention, abnormal ammonia metabolism (e.g. hepatic encephalopathy), azotemia, or the use of some drugs (e.g. phenytoin, valproate, metoclopramide, ceftazidime and others) The respiratory system: examination Chest examination Percussion: To assess density of underlying tissue. Done over different respiratory segments, comparing right and left. Results interpretation Resonance – normal Dullness – increased density Atelectasis, alveolar filling/consolidation, pleural effusion, fibrosis Hyperresonance – decreased density The respiratory system: examination Chest examination Auscultation: detect any abnormal sounds including wheezing, crackles, or stridor. Normal ‘vesicular’ breath sounds have a rustling quality.https ://www.youtube.com/watch?v=xnubmm eDWrw Diminished breath sounds: Pleural effusions, pleural thickening, pneumothorax, bronchial obstruction, asthma, or COPD. Wheeze or Stridor: (see before in  The respiratory system: examination Chest examination Auscultation (contd.): Crackles (‫)الخشخشة‬: caused by re-opening, during inspiration, of small airways which have become occluded during expiration. Fine and late in inspiration if coming from distal air spaces: e.g. pulmonary edema Early inspiratory crackles suggest small airways disease (eg COPD) Crackles disappearing on coughing are insignificant α1-antitrypsin deficiency case study The patient: Age: 35-year-old. Gender: Male. A known case of α1-antitrypsin deficiency who died awaiting a second hepatic transplant after he was found to have cirrhosis due to hepatitis C. This presentation will cover: The patient illness history The diagnostic approach and clinical laboratory tests A summery on the pathophysiology of the Past medical and surgical history The patient disease started to manifest at the age of 6 weeks as: Jaundice which resolved spontaneously at age 2 months. At age 20 months: Enlarged liver Liver biopsy showed: Postnecrotic cirrhosis Presence of globules that were PAS+, diastase resistant. Past medical and surgical history At age 2.5 years On physical examination: protuberant abdomen with a palpable liver & spleen LAB tests showed: A normal hematological picture except for low platelet count. Mildly increased Aspartate transaminase (AST; a liver enzyme). low albumin & alpha1-globulin band. Protease inhibitor phenotyping was done on the child and his family and showed that he was PIZZ, while his both parents had the heterozygote Past medical and surgical history At age 6 year: severe ascites & peripheral edema necessitated the initiation of spironolactone. episodes of peritonitis and sepsis At age 11 years: LAB tests showed: Decreased Crcl. High urinary protein excretion (protein losing nephropathy) Abnormal coagulation studies. Renal biopsy confirmed glomerulonephritis Past medical and surgical history At age 12: chest pain from pneumothorax, which required chest tube insertion. severe airway obstruction on pulmonary function test (consistent with emphysema). confused and disoriented. Lab test shows: elevated ammonia level (consistent with acute hepatic encephalopathy that was controlled with neomycin enemas “Neomycin inhibits ammoniagenic coliform bacteria that  Past medical and surgical history During last admission (age 12): GI bleeding, hepatic coma, and increased intracranial pressure. Treated with hyperventilation, mannitol drip & barbiturate. He recovered and maintained an active life with: limited protein intake, neomycin, and lactulose “Lactulose is a non-absorbable sugar used in the treatment of constipation and hepatic encephalopathy. Lactulose helps trap the ammonia (NH3) in the colon and bind to it. It does this by using gut flora to acidify the  Past medical and surgical history At age 16 years: Lab test showed: Crcl= 23 ml/min ( decreased renal function). accepted as a candidate for a combined liver-kidney transplant. At age 18 years: the transplant was successfully performed. At age 35: hepatitis C (likely acquired from many blood transfusions he required prior to & during his transplant). Cirrhosis was put as a candidate for a liver transplant Diagnosis The patient experienced a complicated clinical course of alpha-1 antitrypsin deficiency: 1. liver disease: cholestatic liver disease. developed into hepatic cirrhosis “confirmed by biopsy”. phenotype PIZZ. all indicate alpha-1 antitrypsin deficiency. 2. Lung disease: Emphysema associated with alpha-1 antitrypsin deficiency develops at early age, as was this patient. Symptoms associated with emphysema: Diagnosis 3. Renal disease: seen in 17% of infants with alpha-1 antitrypsin deficiency (also observed in this patient). Findings of renal disease include: massive proteinuria hypoalbuminemia Renal failure Differential Diagnoses Since many complications may developed in early life in patient with alpha1-antitrypsin deficiency, there will be many differential diagnosis need to be ruled out. Liver disease Autoimmune Hepatitis (liver dysfunction) Viral Hepatitis Cystic Fibrosis Lung disease Primary Ciliary Dyskinesia (kartagener syndrome) Cystic fibrosis Other causes of COPD Clinical laboratory tests 1. Screening by serum protein electrophoresis small or undetectable alpha-1 globulin band in serum protein electrophoresis should lead to more specific diagnostic tests. Clinical laboratory tests 2. A1AT Test: Three types of A1AT tests are available: Serum level: Levels of A1AT protein in blood (alone has a low sensitivity for detecting AATD). Phenotype testing by isoelectric focusing (IEF). PiZZ phenotype responsible for nearly all cases of A1ATD emphysema and liver disease (required to confirm A1ATD). Genotype testing (DNA testing) Clinical laboratory tests Other tests related to disease manifestations: Liver enzymes (Abnormal due to liver dysfunction associated alpha1-antitrypsin deficiency). coagulation studies (Abnormal due to liver dysfunction associated alpha1- antitrypsin deficiency) Urinary Protein excretion (renal disease associated alpha1- antitrypsin deficiency). Ammonia serum level ( liver disease associated alpha1-antitrypsin deficiency). What is α1-Antitrypsin? A glycoprotein produced by hepatocytes. Function: A protease inhibitor, Protect tissues from proteolytic enzymes released during the inflammatory response (it inhibits various proteases (not just trypsin) primarily targeting neutrophil elastase ). Mode of Action: Elastase recognizes α1-antitrypsin as a substrate and attempts to cleave it. Irreversible reaction between α1- antitrypsin and elastase at methionine residue. elastase is trapped with α1-antitrypsin & cleared Pathophysiology Genetically inherited autosomal- codominant Disease Caused by mutations in the SERPINA1 gene which alters the configuration of the alpha1-antitrypsin molecule and prevents its release from hepatocytes. This lead to: Decreased serum levels of alpha1- antitrypsin. Low alveolar alpha1-antitrypsin concentrations, so no protection against proteases. Destruction of alveolar walls (emphysema). Pathophysiology liver and lung damage in AATD http://scitechconnect.elsevie r.com/aat-deficiency-cure-alp has/ Management Treatment of lung disease may include bronchodilators, inhaled steroids, and when infections occur antibiotics. Intravenous infusions of the A1AT protein or in severe disease lung transplantation may also be recommended. In those with severe liver disease liver transplantation may be an option. Avoiding smoking and getting vaccinated for influenza, pneumococcus, and hepatitis is also recommended. IV infusions of alpha-1 antitrypsin is an option for lung disease (not appropriate for people with liver disease), recommended at the onset of emphysema