CM and PARA - Analysis of Body Fluids (Day 5 and 6) PDF

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I. Clinical Microscopy and Parasitology Day 5 and 6: July 12-Topics:  Analysis of Other Body Fluids  Semen AnalysisReferences: Video AUBF 6thGen by StrasingerCEREBROSPINAL FLUID EXAMINATIONCerebrospinal Fluid Examination  Diagnosis of central nervous system (CNS) abnormalities  Samples are obtai...

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CLINICAL INTERNSHIP 1 Day 5 and 6: July 12-13

I. Clinical Microscopy and Parasitology Topics:  Analysis of Other Body Fluids  Semen Analysis

References: Video AUBF 6th Gen by Strasinger →

CEREBROSPINAL FLUID EXAMINATION

Fibrin clots - can be found if the protein contents in the CSF is very high and also possible under traumatic tap condition

Cerebrospinal Fluid Examination  Diagnosis of central nervous system (CNS) abnormalities  Samples are obtained: Lumbar, Cisternal, Lateral cervical puncture, and, Ventricular cannulas or shunts Sample Collection  Typically 3-4 tubes are drawn  Tube 1 = Biochemistry  Tube 2 = Microbiology  Tube 3 = Hematology (cell counts)  Tube 4 = Cytology  Traumatic tap - Tube 3 must be used for maximum discs  Microbiology - Never use Tube 1 (contamination w/ skin bacteria)  Recommendation - 12 ml of CSF partitioned into three to four sterile tubes  It is important that CSF is not allowed to sediment before partitioning Sample Stability  CSF must be analyze immediately after collection (less than 1 hour after collection)  The stability of the CSF samples varied depending on procedures ordered.  Hematologic analysis - performed within one hour of fluid aspiration → RBCs and WBCs have limited stability in CSF (since CSF is hypotonic and cells can rapidly lyse)  Timing is critical for WBCs → Number and type of cells present are clinically important in diagnosing cases of meningitis → Detecting CNS leukemic involvment  CSF samples for hematologic testing should be maintained at room temperature prior to testing Refrigeration is NOT RECOMMENDED for some culture  specimens → Such as Haemophilus influenzae and Neisseria meningitidis may not survive at refrigerated temperatures  Store 3-4 ml of CSF at 4°C for → General investigations → Investigation of bacteria and fungi → Antibody testing → Polymerase chain reaction (PCR) → Antigen detection  Larger volumes (10-15 ml) is necessary for certain pathogens  Mycobacterium tuberculosis  Fungi or Parasites Gross Examination  Color: → Hold the sample against a white paper and compare it to a tube of distilled water  Any color = Abnormal (presence of blood may be due to pathology or trauma during the procedure)  Yellow = Xanthochromia (due to previous hemorrhage and lysis of RBCs 

Clarity → Normal CSF = Clear → Turbidity: Increase in the number of cells

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Grossly Bloody Cerebrospinal Fluid (CSF) → Possible to distinguish if the presence of blood is due to:  Subarachnoid hemorrhage  Traumatic Tap

Procedures for CSF Cell Count  Equipment required  Neubauer chamber  Microscopic slides  Leishman’s stain  Centrifuge 

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Total Count: → Mix the specimen thoroughly by gentle inversion at least 10 times (10x) → Using a pipette transfer the undiluted fluid to hemacytometer counting chamber → Fill both the sides of chamber using proper technique → Allow the cells to settle → Focus under low power (10x) and adjust condenser and diaphragm to maximum visualization → Switch to high power (40x) → For undiluted sample, all 9 squares are counted → Average the results from both sides of the chamber → NOTE: the necessity to dilute the CSF and the number of squares counted depends upon the cellurality of the specimen and adjustment in the procedure should be made accordingly. → Both WBCs and RBCs should be counted. If a diluent is required - Isotonic saline may be used (preserved WBCs and RBCs) Dilution 1:1 (adequate to obtain a cell count) Counts is extremely high - Dilution with Turk’s fluid RBCs and WBCs are counted separately Red Blood Cells (RBCs) → Care should be taken to check if RBCs are crenated or not → Crenated - subarachnoid hemorrhage → Normal - Traumatic tap → Distinguish between neutrophils and lymphocytes and determine the percentage

CLINICAL INTERNSHIP 1 Day 5 and 6: July 12-13

I. Clinical Microscopy and Parasitology Topics:  Analysis of Other Body Fluids  Semen Analysis

References: Video AUBF 6th Gen by Strasinger

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NOTE  Necessity to dilute the CSF and the number of squares depends upon the celluarity of the specimen  Adjustment in the procedure is made accordingly  Both WBC and RBC are counted

Gram stain

Culture

Syphilis serology

RBC count

Negative

Sterile

Negative

No RBC

Calculation: 

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9 = Area counted N = Number of cells 1/10 = Depth factor 1 = Dilution

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Differential WBC Count → Spin the sample at 1500 rpm for 1 minute → Discard the supernatant → Re-suspend the sediments in the available fluid → Make a smear → Add Romanowsky stain (Leishman stain) → Allow to stand for 1 minute → Dilute with buffer of pH 6.8 → Allow to stand for 3 minutes → When dry, view the slide under microscope → Do differential count of WBCs → Quality assurance mechanism of inter-observer variance is done on a periodic basis and that is testing by at least 2 people and analyzing each report should be done in periodic basis

Interferences  Improper mixing before charging  Improper centrifugation  Air bubbles  Sitting time beyond 20 minutes - results disintegration of cells  Stain debris Biochemistry  CSF Protein and Glucose  measured in same way  done in serum samples  CSF - Protein concentration:  Normal = 14 - 45 mg/dL  Derived from the plasma  CSF - Glucose concentration:  Normal = 40 - 80 mg/dL

Gross appearance

Opening pressure

Specific gravity

Glucose

Total protein

Clear and colorless

50-175 mm H2O

1.0061.009

40-80 mg/dL

15-45 mg/dL

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FLUID EXAMINATION - GENERAL Small amounts of serous fluid lies between the serous membranes lining the body cavities and those covering the organs within the cavities. It is an “ultrafiltrate” of the plasma derived from the surrounding capillaries. It is continuously produced depending on → Capillary hydrostatic pressure → Plasma oncotic pressure → Capillary permeability There is also continuous reabsorption of the fluid through surrounding lymphatic’s and venules. An excess accumulation of this fluid is called effusion, which results from an imbalance of production and reabsorption. Examples of these fluids sent to the lab for examination are  Peritoneal fluid (ascitic fluid)  Pleural fluid (thoracic fluid)  Pericardial fluid  Synovial fluid (from the joints)  Cerebrospinal fluid

Body Fluid Examination  Aim of testing the Serous body fluids → Differentiating transudates from exudates - it helps to understand if the underlying cause is systemic or localised → To differentiate inflammatory from non-inflammatory cause like malignancy → To identify specific cause like tubercular etiology 

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Reference Ranges

Leukocytes: → 0-5/ μl = Adults and children → Up to 30/ μl = Newborns Differential counts: → 60-80% lymphocytes → 30% monocytes and macrophages (higher in neonates) → 2% or less other cells → Up to 30/ μl = Newborns

Fluid Examination includes: → Routine examinations  Gross examination  Cell counts  Biochemical examination  Culture, gram stain and cytology → Special examinations  Clinical picture and provisional diagnosis Fluid Sample → All the samples should be received in the laboratory in appropriate containers with the test requisition form giving history and provisional clinical diagnosis. → When fluid sample is received in the laboratory it is sent to different departments for testing.  Hematology - for total and differential cell counts (EDTA tube)  Biochemical - glucose, protein, lactate levels (Plain tube- red or Heparin)  Microbiology - culture and gram staining (sterile container or culture bottle)  Clinical Pathology: Cytology (Plain tubes or Heparin)

CLINICAL INTERNSHIP 1 Day 5 and 6: July 12-13

I. Clinical Microscopy and Parasitology Topics:  Analysis of Other Body Fluids  Semen Analysis

References: Video AUBF 6th Gen by Strasinger Are as per the analyte as described in the biochemistry section and can be run  Semi-automated  Fully automated biochemistry analysers Kit inserts → Should be studied well before deciding to use the method for analysis → For instance, proteins in some fluids like CSF are in very small quantities and require specialized reagents and special calibrators for microproteins assessment Manual Tests → SSA are semiquantitative and not standardized to specify the protein levels in fluids. → Specified sample collection procedure should be followed especially with reference to the container for collection If investigations are not available → Understand the storage and transportation requirements well these may vary as per analyte and method of examination. → This can be understood from the referral laboratory → For example, in the case of LDH, it is better not to refrigerate or freeze specimens, but to hold it at room temperature (15-25 Celsius)  Serum should be separated from clot, within 1 hour, to minimize hemolysis → NOTE: Most biochemical parameters are reported with reference to the corresponding serum values for making clinical decisions.  Eg. Serum protein / Pleural fluid protein  Serum amylase/ ascitic fluid amylase  Pleural fluid / serum LDH ratio  Serum-Ascites Albumin Gradient (SAAG)  Therefore, it is important to analyse the serum parameters simultaneously, these points are dealt with later in the section

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Sometimes the sample may be sent to the lab in a syringe. → In that case distribute the sample into the required tubes, depending upon the volume. → Low volume sample - can be accepted in a single container and tests should be carried out by different departments using the same sample container.

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Inoculate culture plates > Biochemistry tests > Cell counts > centrifuge sample > Cytology 

Sample Collection:  All samples are collected under sterile conditions by an experienced doctor  Pleural Fluid → Fluid collected by thoracocentesis for diagnostic and therapeutic purposes  Peritoneal Fluid (Ascitic fluid) → Sample collected through paracentesis for diagnostic and therapeutic purposes  Pericardial Fluid → Sample collected by pericadiocentesis  Synovial Fluid → Collected by arthrocentesis  Cerebrospinal Fluid → Collected by lumbar puncture, which is discussed separately Gross Examination  Volume → Should be recorded when received in the laboratory  Color → Place the fluid in a clear glass tube and look against a white background (Note down the color of the fluid)  Clarity → Can be assessed by placing the tube with fluid against a news print. (Clear, Slightly cloudy, Cloudy, Turbid) → Further quantified as Mild, Moderate, and Marked → Presence or absence of coagulum → Cloudy or purulent fluid - most often associated with an inflammatory process → Hemorrhagic fluid - Might indicate  Traumatic tap  Malignant neoplasm  Infarction or trauma → Will appear turbid or milky, even after centrifugation → Pseudochylous - Effusions may be milky or greenish and have a sparkly sheen from the accumulation of cholesterol crystals. When the fluid is clear to strawcolored, further workup may be unnecessary unless chemical examinations indicate an exudative process. Biochemical Investigations  Total Proteins  Glucose  Lactate  Enzymes  Lactate dehydrogenase  Adenosine deaminase (ADA)  Amylase  Parameters requested → Depend on the type of fluid and clinical diagnosis → NOTE: If the sample is very turbid or cloudy, centrifuge the sample and then test using the supernatant.  Investigations:

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Cell Counts  Cell count should be performed promptly within 4 hours as the cells begin to degenerate  Count are usually performed manually using Neubauer chamber  If the fluid is clear and low cell count is suspected, do not dilute and do a direct count.  If the fluid is turbid or hemorrhagic and high cell count is suspected cell count is done after appopriate dilution with saline  Automated cell counter may also be used for high cell counts  Aperture of the machine can get clogged and it could also result in falsely high counts due to non-leukocyte content like crystals, fat globules or mesothelial cells. Cytological Examination  Fluid is processed to prepare smears for microscopic examination of  Types of cells  Background material  The fluid received is stirred briskly to disperse the suspended cells  If the fibrin clot has already formed, the clot may be smashed against the sides of the tube to free the trapped cells.  Centrifugation

CLINICAL INTERNSHIP 1 Day 5 and 6: July 12-13

I. Clinical Microscopy and Parasitology Topics:  Analysis of Other Body Fluids  Semen Analysis A representative volume of the fluid (10-15 ml) is usually centrifuged at 2000 rpm for 10 minutes in a calibrated centrifuge Smears are then prepared from these deposits for cytological examination and gram staining Cytocentrifuge - it concentrates small number of cells suspended in fluid specimens at 2000 rpm for 2 minutes and sediments cells directly to slides. → The filter card simultaneously absorbs the fluid medium → The result is a mono layer of well-preserved cells with in an area of 6mm. All the prepared slides should be fixed adequately in ethanol/methanol/spray fixed → Ethanol Fixation - Wet fixation is done by immediately immersing the prepared slides into ethanol in Koplin jars.  Some stains like MGG, the slide may be air dried.  Later wet fixed in methyl-alcohol for 15-20 minutes during staining process.  Alternatively, spray fixation can be done using cytosprays Smears are stained using  Giemsa  Leishman  MGG or PAP stain →

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Microbiological Investigation  Culture and Sensitivity → Ideally the sample should be inoculated into the culture bottle at the bedside → If not possible it should be processed in the microbiology department in the laboratory → Staining of centrifuged deposit for gram stain and AFB Special Tests  These tests are done for specific conditions if indicated clinically  Include: Immunological tests and Tumor Markers FLUID EXAMINATION - SPECIFIC Pleural Fluid Examination  Routine tests → Gross Examination → Chemical Examination  Protein  Glucoce  Pleural fluid/serum protein ratio  Pleural fluid/serum LD ratio  Cholesterol level → Cell Count  Total and differential → Cytology → Culture and Gram Stain  Additional tests → Enzymes: Amylase and LDH → Lipids: Chylous and Effusions → Tumor markers: CEA → Immunological: RA and ANA factor, Complement levels  Normal Pleural Fluid → Normal pleural fluid is a transudate and has the following characteristics → Clear ultra filtrate of plasma that originates from the parietal pleura

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References: Video AUBF 6th Gen by Strasinger pH Protein WBC Glucose LDH 

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7.60 - 7.64 1000/μL  Suggest exudate  Infective etiology → Differential with Neutrophils >50%  Bacterial pneumonia  Pulmunary infarction  Early tuberculosis → Differential with Lymphocytes >50%  Tubercular  Viral  Malignancy  Rheumatoid pleuritis  SLE → Differential with Eosinophils >10%  Pulmonary infarct  Trauma  Rheumatological disease → RBC count > 100,000/μL  Malignancy  Trauma  Pulmonary infarct Cytology → Mesothelial cells are reactive cells common in pleural fluid which are shed by the pleura. Seen in  Inflammatory  Rheumatoid

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Interpretation of Pericardial Fluid  Gross Appearance → Large volume (>350 ml) seen in malignancy or uremia → Turbid fluid suggests  Infection or malignancy → Blood like fluid  Hemorrhagic effusion  Blood from heart chamber - check the hematocrit, it will be the same as that of venous blood with clot 

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Lupus erythromatous cells (SLE) may be seen in some cases of effusion from SLE

Pericardial Fluid Examination  Normally 10-50 mL of fluid is present in the pericardial space around the heart.  The process of fluid formation is same as described earlier  Recommended test for pericardial effusion are:  Routine tests: → Gross Examination → Chemical Examination - Protein and Glucose → Cell count

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→ Cytology → Culture and Gram stain Additional tests → Enzymes - ADA for tubercular pericaditis → Lipids - Chylous effusion and Pseudochylous effusions

Chemical Examination → Protein levels have little use → Glucose level 10,000/μL suggest  Bacterial  Tuberculous or malignant pericarditis Cytology → The PAP or Giemsa stained smears may show presence of malignant cells, commonest being metastatic cells from breast or lung malignancy.

Ascitic Fluid Examination  Normal peritoneal fluid is less than 50 ml of transudative fluid in the mesothelial lined peritoneal cavity, which is ultrafiltrate of plasma across capillary walls  When there is excessive accumulation of free fluid in peritoneal cavity it is called ASCITIS  Fluid received in the laboratory is called ASCITIC FLUID  Common causes for Ascitis: → Transudative  Congestive cardiac failure  Cirrhosis  Nephrotic syndrome → Exudative  Infections  Malignancies  Traumatic  Ammonia levels  Classification into transudates and exudates is not well defined for ascitic fluid  Sample - Ideally 50-100 ml of fluid should be sent for examination

CLINICAL INTERNSHIP 1 Day 5 and 6: July 12-13

I. Clinical Microscopy and Parasitology Topics:  Analysis of Other Body Fluids  Semen Analysis 

References: Video AUBF 6th Gen by Strasinger

Recommended test for ascitic fluid include: Routine tests: → Gross Examination → Chemical Examination  Protein  Glucose  Serum ascitis albumin concentration gradient (SAAG) → Cell count - total and differential count → Cytology → Cultute and Gram Stain  Additional tests: → Alkaline phosphatase → Lactate dehydrogenase (Malignant diffusions) → Creatinine and urea (Differentiate peritoneal fluid to urine) → Ammonia levels (Perforated, ruptured appendix)

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Interpreting Ascitic Fluid  Gross Examination → Transudate:  Pale yellow  Clear fluid → Exudate:  Cloudy or turbid fluid  Leukocytes or tumor cells → Green yellow colour could be due to perforation of GI tract or biliary tract → Blood tinged or grossly bloody fluid  Traumatic tap  Malignancy  Tuberculosis  Intraabdominal organ rupture 

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Cell Count → There is no “standardized” ascitic fluid cell count → Generally accepted “cut-off” limits of normal is less than 250 PMNs/mm3 → Cell count of >500 cells/cumm with 70% PMNs is highly suggestive of spontaneous bacterial peritonitis → In tuberculous ascites there is a lymphocytic predominance Chemical Examination → Serum-Ascites Albumin Gradient (SAAG) - superior to total protein concentration to differentiate cirrhosis as a cause  Calculated as: Serum albumin - Ascitic fluid = Serum albumin/Ascitic Gradient → Low SAAG ( 10U/L suggests  Hollow visceral injury  Also helps differentiate between primary and secondary bacterial peritonitis LD activity is often increased in malignant effusion An ascitic fluid/serum LD ratio greater than 0.6 ADA levels are used to identify patients with tuberculosis in endemic areas using a cut-off of 30 U/L Tumor markers like CEA, CA-125, CA 19-9 used to differentiate different intraabdominal tumors

Culture and Gram Stains → Cultures should be obtained by inoculating blood culture bottles at the bedside → This has been shown to improve sensitivity to at least 80%, compared with 50% for “conventional” culture methods → Gram...