Week 15: Synovial Fluid & Sweat Analysis PDF

AUBF311: ANALYSIS OF URINE AND BODY FLUIDS (LECTURE) WEEK 15: SYNOVIAL FLUID & SWEAT ANALYSIS 1ST SEMESTER | S.Y 2023-2024 Instructor: PROF. JUSTIN KIM CABACTULAN VERGARA, RMT, MPH SYNOVIAL FLUID...

AUBF311: ANALYSIS OF URINE AND BODY FLUIDS (LECTURE) WEEK 15: SYNOVIAL FLUID & SWEAT ANALYSIS 1ST SEMESTER | S.Y 2023-2024 Instructor: PROF. JUSTIN KIM CABACTULAN VERGARA, RMT, MPH SYNOVIAL FLUID Different Parts of Joints Synovial fluid analysis is also known as “joint fluid analysis”, in which the main purpose is to diagnose the cause of joint inflammation* Most of the time, joint fluid is submitted/delivered in the laboratory for fluid analysis, especially if the patient has inflammation on his/her joints* Each joint of the human body contains synovial fluid* It is a very thick fluid/liquid, which is used to lubricate the joints and it allows ease of movements* In joint diseases, such as arthritis, that is the time that the joints produce a lot of synovial fluid* The articular cartilage in the joint is avascular, which means If there’s an accumulation of synovial fluid, it means that that it has no blood vessels; there’s no blood in this particular there’s a problem with your joint tissues or within your cartilage* joints; there’s inflammation in that area* In chicken, the cartilage is the white part on both ends of the Synovial fluid is from the term “synovial” or “synovium”, chicken bone * which means egg in Latin* Any damage or inflammation in the synovial membrane will Viscosity resembles the egg white cause pain and stiffness in the joints; it is collectively called as Viscous fluid circulating in the diarthroses is called as arthritis* synovial fluid Arthritis is when there is damage in the synovial membrane* All of the movable joints contain synovial fluid* It is like an oil that lubricates the joints* Functions of Synovial Fluid There’s friction, which can cause damage and then later on 1. Lubricates the joints be inflamed* 2. Reduce friction between joints Hyaluronic acid is a component of synovial fluid which is 3. Provides nutrients to the articular cartilage responsible in the viscosity or the thickness of the synovial 4. Lessen the shock of joint compression occurring during fluid* activities such as walking and jogging Hyaluronic acid is produced by the cells that surround the Synovial fluid keeps the bones slightly apart* synovial fluid called synoviocytes* o The bones should not be touching because it will Synovial fluid is also considered an “ultrafiltrate of cause friction* plasma” with the combination of hyaluronic acid* o The cartilage covering will be protected from wear The synovium, where you can find the synovial fluid, is a and tear* tissue lining in the synovial tendon, which is usually seen in o We need to prevent the damage on the cartilage the diarthrodial joints or with the movable joints* coverings* The plasma is being filtered in synovium o It protects your cartilage* The filtration is nonselective usually except for high- molecular-weight proteins. It lubricates the joints* o When it comes to other compositions, as long as they o It helps the joints to work freely and easily* can pass through the membrane, they can go inside the synovial fluid Provides nutrients to the articular cartilage Synovial Fluid o Since it is an ultrafiltrate of plasma, it also provides - A.K.A joint fluid nutrients for the articular cartilage* - Synovial = Latin word for egg o The analytes coming from the plasma could pass - Viscous fluid circulating in the diarthroses (movable joints) through the membrane and they will supply nutrients on - Viscosity is due to hyaluronic acid produced by that area since some parts of the joints are synoviocytes avascular* - An ultrafiltrate of plasma across the synovial membrane. The filtration is nonselective except for the exclusion of Lessen the shock of joint compression occurring during high-molecular-weight proteins activities such as walking and jogging o During activities, it is like a shock absorber o Like a shock absorber, it lessens the shock of joint compression during activities, such as walking and running* Specimen Collection Method of collection = arthrocentesis Normal synovial fluid does not clot Diseased joints may clot For example: If a patient has an accumulation of synovial fluid, the doctor would usually request for analysis. They will aspirate synovial fluid directly from the joint through arthrocentesis* Arthrocentesis: a very invasive procedure because usually the doctors do not give any anesthesia, not even topical anesthesia* o They will insert a needle in between the joints* o Also known as joint aspiration* o Invasive since it requires a needle; it is through needle aspiration* Page 1 of 7  o For example, there’s an inflammation due to fluid accumulation from the affected joint, they will aspirate fluid using the needle and syringe* Volume Normal = less than 3.5 mL Inflammation = more than 25 mL B. Clarity: Normal: transparent or clear Turbid: due to wbc, crystals, fibrin, or free-floating rice bodies Opaque, milky: crystals Ground pepper: ochronosis Normally, there’s only a very small volume of synovial fluid in Oily, shimmering: RCM the joint. However, during inflammation, or even during infection, expect that the fluid that accumulates in the For clarity, you have to check whether you have a turbid or a synovium will increase; during inflammation, there’s a transparent (normal; no formed elements)* higher amount of fluid* After receiving the specimen, you have to check it, primarily If you observed a turbid color, it may be due to cells (e.g., the physical examination: color, clarity, and viscosity* WBC), crystals, fibrin, and sometimes free-floating rice 1 mL is enough for laboratory analysis* bodies* o Free-floating rice bodies are made up of collagen that are normally seen in skin and joints. The collagen is Distribution covered with fibrinous tissue. They may resemble 1. Microbiology = Sodium heparin or SPS shiny grains of rice. They are usually encountered in 2. Hematology = Sodium heparin / liquid EDTA patients with rheumatoid arthritis, which is an 3. Chemistry and other tests: nonanticoagulated autoimmune disorder* 4. Glucose analysis: sodium fluoride Opaque, milky: due to crystals* If you want to test the synovial fluid, you have to use anticoagulants* Most Common Encountered Crystals: Gram staining and Culture: you have to use Sodium 1. Monosodium urate (MSU) crystals: it causes gout or gouty heparin or SPS, which are used for bacteriological analysis* arthritis* Hematology: Sodium heparin / liquid EDTA; if you have to 2. Calcium pyrophosphate dihydrate (CPPD): it can cause count the cells* pseudogout* o Cell counting is also performed for fluid analysis* Chemistry: Nonanticoagulated tubes Ground pepper appearance: is actually ochronotic shards, Glucose Analysis: Sodium fluoride to preserve the glucose which causes ochronosis or ochronotic arthropathy* present in the joint fluid* o Degeneration of the cartilage occurs* Never use powdered anticoagulants since it may interfere o Those pigmented cartilages were produced due to the with crystal identification* degeneration of the synovium* o Crystals that may be present are one of the things you o There are debris from joint degeneration* have to check using synovial fluid* o Sometimes, the accumulation of these crystals may Oily or shimmery appearance: is due to radiographic induce inflammation* contrast media (RCM) especially in patients undergo MRI* o The use of powdered anticoagulant can definitely o Those magnets will go to the smallest organ to check affect the results, especially during microscopic for any blockage within the organ; this is how we use the examination or crystal examination* RCM for MRI* o Never use lithium heparin, use sodium heparin C. Viscosity: instead* Normal A. Color: o Able to form a string (4-6 cm long) o Ropes / Mucin Clot Test (Hyaluronate Polymerization For physical examination, you have to check the color and Test) clarity first* Normally, you will see a colorless to pale yellow color since it is just an ultrafiltrate of plasma* Test - Ropes/Mucin Clot Test (Hyaluronate Polymerization Test) Deeper yellow is due to inflammation* Reagent: 2-5% acetic acid Greenish tinge is due to bacteria that causes septic arthritis, which is an invasive bacterial infection* Grading Appearance Red: traumatic tap during collection can introduce blood to Good Solid Clot the specimen; or hemorrhagic arthritis* Fair Soft Clot Appearance Significance Low Friable Clot Colorless to pale Normal Poor No clot yellow Deeper Yellow Inflammation We perform a string test* Greenish tinge Bacterial Infection Normally, the synovial fluid can form a string with a length of Red Traumatic Tap, hemorrhagic arthritis 4-6 cm* Milky (+) Crystals It is important to test the synovial fluid because as time passes, it can lose its viscosity* Once you have received the specimen, immediately perform the tests required* Page 2 of 7  Ropes/Mucin Clot Test* For differential count, we have to use cytocentrifuge; we o It is used to check for the formation of clot* are going to perform cytocentrifugation or prepare thinly o 2-5% acetic acid is used; with the addition of this, smear slides and incubate it with hyaluronidase prior to there should be production of solid clot, which should preparation to decrease the viscosity during centrifugation; be surrounded by a clear fluid (normal)* Add hyaluronidase before you centrifuge your sample* Sometimes, there are some bacteria that produces the enzyme hyaluronidase, which degrades the hyaluronic Neutrophils - 20% acid* Elevated in: Urate Gout, Rheumatoid Arthritis (Septic o Hyaluronic acid is used for the viscosity to the Arthritis and Inflammatory Disorders) synovial cells; it is produced by the synovial cells for Most predominant in polymorphonuclear cells* the fluid to be viscous* Higher than 20% is caused by invasive infection, sometimes due o If you have hyaluronidase-producing bacteria, it may to septic arthritis* inhibit hyaluronic acid and further prevent the The neutrophils will increase in the presence of gout and formation of clot or string = it causes false-negative rheumatoid arthritis* reaction* o If the sample contains bacteria such as Lymphocytes - 15% Staphylococcus aureus, Streptococcus pyogenes, and Elevated in: Early Rheumatoid Arthritis, Chronic Infection, Clostridium perfringens, which all contain the Collagen Disorders (Non-septic Arthritis) hyaluronidase enzyme, especially the S. aureus, it will Usually seen only in early rheumatoid arthritis; not produce a clot* It also increases in case of viral arthritis, chronic infection, and non-septic arthritis* Neutrophil also contains hyaluronidase o High presence of neutrophils can inhibit the hyaluronic Monocytes - 65% acid and further prevent the formation of clot or Elevated in: Viral Arthritis, Serum Sickness, SLE string* Most predominant in PMN cells is neutrophil* CELL COUNT Most of the time, monocyte will increase if there’s a problem with the antibodies or autoantibodies (you have autoimmune WBC COUNT disease)* Most frequently performed count Diluting Fluids: Eosinophils - 2% 1. NSS with methylene blue Elevated in: Rheumatic Fever, Metastatic Carcinoma, Lyme 2. Hypotonic solution Disease, Parasitic Infection, Chronic Urticaria 3. Saline with saponin Least amount* ▪ Hypotonic solution and Saline with saponin are Increase in allergy or severe allergy and sometimes due to used if you have RBC contamination or a lot of RBCs carcinoma in patients with osteosarcoma* in the sample; it is for the lysis of RBCs. Some of the cells will also go in the synovial fluid* For Very Viscous Fluid: 1. Add a pinch of hyaluronidase to 0.5 mL of fluid DIFFERENTIAL COUNT 2. Add 1 drop of 0.05% hyaluronidase in phosphate buffer Cell Normal Value per mL of fluid (incubate at 37°C for 5 minutes) RBC Less than 2,000 /uL WBC 150-200/uL* WBC Differential 65% = Monocytes and Macrophages Less than 25% = Neutrophil Less than 15% Lymphocytes and Eosinophils If you have observed cells, for example, cells that are more than We usually count for WBC to check for septic arthritis* 100,000, either RBC or WBC, it indicates severe infection, septic If you have infection, mostly we have S. aureus* arthritis, or inflammatory arthritis* Make sure to lyse the RBCs present in the sample before performing the WBC count* CELLS AND INCLUSIONS SEEN IN SYNOVIAL FLUID Diluting fluids: Hypotonic solution and Saline with Cell / Inclusion Description Significance saponin are used if you have RBC contamination or a lot of Neutrophil Polymorphonuclear > Bacterial sepsis RBCs in the sample; it is for the lysis of RBCs. We have to leukocyte > Crystal-induced count the WBCs (only) that’s why we have to lyse the RBCs* inflammation Never use acetic acid for joint analysis because acetic acid induces clot, which may cause a false increase in Lymphocyte Mononuclear > Nonseptic viscosity. Too much viscosity is considered abnormal* leukocyte inflammation If you don’t have a lot of RBCs in the sample, you can use NSS with methylene blue. The methylene blue will stain the Macrophage Large mononuclear > Normal WBC* (monocyte) leukocyte, may be > Viral infection vacuolated For Very Viscous Fluid: You don’t have to use other enzyme if the sample is highly Synovial lining cell Similar to > Normal viscous or clotted, you can only use hyaluronidase that macrophage, but > Disruption from dissolves the hyaluronic acid to decrease the viscosity* may be multinucle- arthrocentesis You can’t analyze highly viscous fluid, you have to decrease ated, resembling a the viscosity first (using hyaluronidase enzyme)* mesothelial cell DIFFERENTIAL COUNT >Support the lining You also have to perform differential count to picture out the of synovium** cause of inflammation* Page 3 of 7  LE cell Neutrophil > Lupus Crystal identification is a very important diagnostic containing erythematosus (LE) procedure and part of arthrocentesis* characteristic Usually, if the patient has an inflamed joint, there’s a ingested "round possibility that it is due to crystals or bacteria* body" To differentiate the two, you have to do arthrocentesis* If you happen to find a crystal, you have to identify the Reiter cell Vacuolated > Reactive arthritis composition of that crystal* macrophage with (infection in another Most of the time, the accumulation of crystals will lead to ingested part of the body) inflammation; WBC will also increase* neutrophils Most of the time, if there’s crystal-induced arthritis, neutrophils and macrophage will increase* RA cell (Ragocyte) Neutrophil with > Rheumatoid dark cytoplasmic arthritis Causes on why these crystals accumulate in the joint fluid: granules containing > Immunologic 1. Metabolic Disorders. immune inflammation For example, you have increased purine metabolism, your complexes uric acid will also increase; ↑ purine metabolism = ↑ uric acid* Cartilage cells Large, > Osteoarthritis Uric acid will be excreted in the urine, and some uric acid Rice bodies multinucleated will be deposited in the joint, specifically in the joint fluid in cells the form of MSUD or Monosodium Urate* For example, if the patient has problem with the breakdown Rice Bodies Macroscopically > Tuberculosis of uric acid – for example, the patient has Lesch-Nyhan resemble polished > Septic syndrome, aside from the urine, uric acid crystals can also rice > Rheumatoid be seen in the joints. The patient does not have the enzyme Microscopically arthritis necessary to breakdown or metabolize the uric acid. show collagen and fibrin 2. Decreased renal excretion that produce increased blood levels of crystallizing chemicals Fat droplets Refractile > Traumatic injury The kidney is not working well so there is a tendency that the intracellular and > Chronic uric acid crystal will accumulate in the blood and be extracellular inflammation deposited into the joints. globules Stain with Sudan 3. Degeneration of cartilage and bones dyes If there’s degeneration of cartilage and bones, it can also produce crystals. Hemosiderin Inclusions within > Pigmented clusters of synovial villonodular 4. Injection of medication (corticosteroid) cells synovitis The patient can accumulate crystals also because of certain medications (corticosteroid) Neutrophil – most predominant granulated cell* Synovial lining cell – similar to macrophages, but they are there Different Types of Crystals You Might Encounter* to support the lining of the synovium* Monosodium Urate: needle-shaped rods LE cell – LE stands for Lupus erythematosus because they are o Most common cause of gout* seen in patients with LE* o 90% of acute urate gout is caused by monosodium Reiter cell – for example, the neutrophils contain bacteria and urate. Occasionally, because of septic arthritis as well* they haven’t burst yet, the macrophage will engulf the infected neutrophil* Calcium Pyrophosphate Dihydrate (CPPD): rhomboids, rod or RA cell – seen in rheumatoid arthritis, which is an autoimmune rectangular disease; therefore, it is expected that there will be an o Associated with degenerative arthritis. * accumulation of immune complexes, or antigen-antibody complexes* Hydroxyapatite & Basic Calcium Phosphate (BCP): small Fat Droplets and Hemosiderin – pigmented cells or clusters needles (apatite) of synovial cells with pigment* o Seen in apatite gout and osteoarthritis. Calcium Oxalate: bipyramidal envelopes o Seen if the patient has renal disorder or is undergoing dialysis. You will also see oxalate gout.* Cholesterol Crystals: notched rhombic plates Corticosteroid: flat, variable-shaped plates o Accumulation of drugs in the synovial fluid. Lipid Crystals: maltese cross Most of the time, the accumulation of crystals causes gout, which is very painful. It is caused by crystal deposition in the articular tissue. And so, the body will respond to it, causing inflammation Crystal Identification (inflammatory response), which will lead to gouty arthritis.* Causes of Crystal Information 1. Metabolic Disorders 2. Decreased renal excretion that produce increased blood levels These types of crystals can be differentiated by using a special of crystallizing chemicals microscope = polarizing microscope 3. Degeneration of cartilage and bones 4. Injection of medication (corticosteroid) Page 4 of 7 Polarizing Microscope Detects for the presence or absence of birefringence Compensated Polarizing Microscope Confirms the type of birefringence (positive or negative birefringence) Red compensator is placed between crystal and analyzer Two of the most commonly encountered crystals: 1. MSU = parallel = yellow = negative birefringence 2. CPPD = opposite = blue = positive birefringence ***picture above are example of MSU Crystals under polarize light CHEMICAL EXAMINATION GLUCOSE: Normal: < 10mg/dL ↑ in infection, inflammation Done in conjunction with the blood glucose* Requires 8 hour fasting* Blood glucose – synovial fluid glucose (subtract) o destroy cells Significance Traumatic injuries (the physician caused traumatic tap), coagulation Deficiencies LABORATORY FINDINGS IN JOINT DISORDERS Color and Clarity Cloudy, red fluid Grouped into 5 to differentiate lab findings in joint disorders. Viscosity Low GROUP I IIa WBC Count Equal to blood Neutrophils Equal to blood Non-inflammatory (Immunologic) The components of the blood also Significance Degenerative joint Immunologic go to the synovial fluid, indicating disorders disorders (RA, SLE) leakage* (osteoarthritis) Glucose Normal (Autoimmune) Not that invasive as compared to others* Others (+) RBCs Color and Clear, yellow fluid Cloudy, yellow fluid Clarity SWEAT ANALYSIS Viscosity Good Poor Examination of Sweat Hyaluronic Measurement of sweat electrolytes (sodium and chloride) is production is performed to confirm Cystic Fibrosis (those with this have affected Ig of cell high level of chloride in their sweat) destruction by complement * Cystic Fibrosis o Metabolic disease that affects the mucus secreting WBC Count < 1,000 /uL 2,000-75,000/ uL glands of the body. (slightly increased*) o Mucus build up in the lungs and other organs.* Neutrophils < 30% > 50% Sweat analysis is not a routine test in the laboratory. It is a (increased) special test being done in reference laboratory.* Glucose Normal (similar to Decreased Why is there elevation of sodium and chloride? blood glucose) o Because of the sweat glands’ inability to reabsorb them. Others (+)Autoantibodies Normally, to maintain homeostasis in the body, we must reabsorb these analytes. GROUP IIb III Cystic Fibrosis Common Indicators 1. Family history (Crystal-Induced) Septic 2. Newborns with intestinal obstruction Significance Gout (MSU) Microbial Infection o In the intestine, there is a problem in the goblet cells or the mucus secreting cells* Pseudogout (CPPD) 3. Appearance of pancreatic insufficiency in infants 4. Respiratory distress in infants Page 6 of 7  o Decreased mucus in the lungs of the newborn* 5. Confirmed by elevated sweat Na and Cl Signs and Symptoms Noticeable salty sweat o Normal to taste salty, but not as salty as the pure form of NaCl* Frequent respiratory infections and chronic cough o Decreased lung mucus = decreased lung surfactants* Bulky offensive greasy stool o Increased fats* Malnutrition Male infertility (obstructive azoospermia) Na and Cl over 70 mEq/L o Consistent in 98% of patient with cystic fibrosis Na and Cl of 40 mEq/L- borderline o Na - Flame photometry –Yellow sodium - Ion exchange chromatography – Na is (+) charged and Cl is (-) charges, you can separate them* o Cl - Titration (manual or automated) - If you have more Cl in sweat, there is more electrical voltage generated in the censor surface. This is why we apply electricity during pilocarpine iontophoresis* Gibson and Cooke Pilocarpine iontophoresis Technique o Actually, this pilocarpine is not a test for sweat, this is just a method of collection. It is used to induce sweat production * o Pilocarpine + Mild current = Sweat production* Test for Chloride in Sweat 1. Cotlove Chloridometer 2. Microtitration with Mercuric Nitrate 3. Cystic Fibrosis Analyzer 4. Chloride Electrode Method More automated, all of them are used to titrate the amount of Cl in the sample. Page 7 of 7