BODY FLUID ANALYSIS FOR CELLULAR COMPOSITION Afsaneh Rajabiani, APCP Tehran Medical University Shariati Hospital

Morphology Assessment of Body Fluid Wedge smears (push smears) should not be used with fluids because of their inferior ability in preserving intact cells. The cytocentrifuge preparation is recommended for air-dried body fluid slides, because: Concentrate the cells Minimizes cell distortion Produces a monolayer of cells Romanowsky-type stained slides show excellent morphologic detail Cell typically are randomly dispersed in a small circular area When malignancy is suspected, the whole cellular area should be evaluated

Morpholoy Assessment of Body Fluid Cells are concentrated approximately 20-fold by cytocentrifugation *Even hypocellular samples with a chamber cell count of zero can have a yield of approximately 35 cells per slide. The quantitative yield, however, varies from 30 to 75%, and smaller cells, such as lymphocytes, may be underrepresentated The speed and time of centrifugation, the amount of sample in the chamber, and the filter paper absorbance are factors that can influence both the cell yield and morophology

Morphology Assessment of Body Fluid Fresh, unfixed specimens should be used, particularly in body fluid samples with low protein contents (such as CSF) Prolonged delay in preparing slides (more than 8 hours), the report should include a statement that the differential count may be inaccurate, due to cellular degeneration Body fluids may contain fibrin and other proteins that can clog the filter card Washing the cells by centrifuging the sample and resuspending in saline, improve the cell yeild and morphology

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CSF CSF collects wastes, circulates nutrients and cushions and lubricate the CNS CSF is produced at a rate of approximately 500ml/day, about 70% derived by ultrafiltration and secretion through the choroid plexus; The remainder by ependymal lining of ventricles and cerebral subarachnoid space Resorption occurs at arachnoidal villi, predominantly along the superior sagittal sinus Total CSF Vol : 90-150 ml in adults and 10-60 ml in neonates In adults up to 20 ml CSF can be removed

CSF Specimen divided into 3 sterile tubes: 1. chemistry & immunologic studies 2. microbiological examination 3. cell count and differential Refrigeration is recommended except for culture specimens because fastidious organisms like H.influenza and N.meningitidis will not survive Indications for LP: 1. Meningeal infection (most important indication) 2. subarachnoid hemorrhage 3. CNS hemorrhage 4. demyelinating diseases

CSF Normal CSF is clear and colorless, with a viscosity similar to water Turbidity or cloudiness begins to appear with WBC>200/ul or RBC>400/ul Other causes of cloudiness: Microorganisms, radiographic contrast material, aspiration of epidural fat and increased protein levels Clot formation seen in traumatic taps, spinal fluid block, suppurative and Tb meningitis Clot not seen in subarachnoidal hemorrhage Viscous CSF seen in metastatic adenocarcinoma, cryptococcal meningitis or attributed to nucleus pulposus Pink-red CSF usually indicates presence of blood Grossly bloody CSF with RBC>6000/ul

CSF Xanthochromia: pink, orange or yellow CSF due to RBC lysis and Hb breakdown Peak density occurs at about 24-36 hours after subarachnoid hemorrhage and gradually disappearing in 4-8 days Visible CSF xanthochromia: 1)RBC lysis 2)Bilirubin 3)CSFprotein>150mg/dl 4)Carotenoids 5)Melanin(brownish CSF) 6)Rifampin

CSF CSF normal WBC count in adults:0-5/ul RBC count in CSF have limited diagnostic value but may allow a useful approximation of true WBC or total protein One WBC added for every 700 RBC 8mg/dl protein for every 10.000 RBC

CSF Ependymal cells and choroid plexus cells may rarely seen The CSF contains few lymphocytes and monocytes 7% neutrophils with a normal WBC count is acceptable Viral induced neutrophilia usually changes to a lymphocytes within 2-3 days Peristant neutrophilic menngitis may be noninfectious or less commonly due to Nocardia, Actinomyses and Zygomycetes Lymphocytes seen in viral,Tb or parasitic meningitis and degenerative disorders Plasma cells are not normally present in the CSF

CSF Mild eosinophilia (1-4%) as a part of a general inflammatory response Parasitic invasion of the CNS is the most common cause of eosinophilic meningitis (Eos>10%) Increased monocytes lack specificity and are usually part of a “Mixed cell reaction”; if without neutrophils is charactristic of viral and syphilitic meningoencephalitis Erythropheges appears 12-48 h following SAH or traumatic tap Hemosiderin-laden macrophages begin to appear after 48 h CSF examination has following sensitivities: Leukemic patients (70%), Metastatic Ca (20-60%), CNS malignancies (30%)

CSF Over 80% of CSF protein is derived from plasma (1% of blood level) Protein concentration increases caudally, Mean lumbar value ranges between 15-45 mg/dl Upper limit of normal for neonates is 150 mg/dl and as high as 400mg/dl in premature infants CSF glucose is normally 50-80 mg/dl (60% of plasma level) Below 40mg/dl is abnormal and caused by anaerobic glycolysis by brain tissue and leukocytes and impaired transport into CSF Glucose level normalize before protein and cell count, making it a useful parameter in response to treatment Increased CSF glucose level is of no clinical significance, only reflecting increased blood glucose level

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Serousal Fluids The closed cavities of body are lined by serosal membranes (pleura – pericardium and pertoneum) The fluid is a plasma filtrate from capillaries of the parietal membrane The fluid is reabsorbed through the lymphatics and venules of the visceral membrane The small amounts of fluid facilitates movement of two membranes The serosal fluids are plasma ultra filtration and mesothelial lining does not add any substance For laboratory assessment needle aspiration is done (Thoracocentesis – Pericardiocentesis – Paracentesis)

Serousal Fluids Transudate and Exudate Evaluation of serous fluids directed first toward differentiating transudate from exudate Transudative effusions (usually bilateral in pleura) have mechanical process owning to systemic conditions, leading to increase capillary hydrostatic pressure or decreased plasma oncotic pressure Exudative effusions (usually unilateral in pleura) have inflammatory process, associated with disorders of vascular permeability or interfere with lymphatic resorption

Serousal Fluids Transudate Exudate Appearance Clear Cloudy Specific gravity < 1.015 > 1.015 Total protein < 3.0 gr/dl > 3.0 gr/dl F/S protein ratio < 0.5 > 0.5 LD < 200 IU > 200 IU F/S LD ratio < 0.6 > 0.6 Cell count < 1000/ul > 1000/ul Spontaneous clotting No Yes Total leukocyte and red cells counts are of limited use in the evaluation of serousal Fluids

Serousal Fluids Pleural Fluid Transudates generally require no further work-up additional testing for cholestrol and albumin gradient may discriminate effusions with equivocal Light’s criteria (the first three criteria) PF/S protein ratio > 0.5 PF/S LD ratio > 0.6 Pleural Fluid LD > 2/3 upper limit of serum Pleural Fluid cholestrol > 45 mg/dl PF/S cholestrol ratio >0.3 Serum-pleural fluid albumin gradient < 1.2 g/dl PF/S bilirubin ratio > 0.6 * Bilirubin measurement has not help as a strong discreminator

Serousal Fluids Pleural Fluid Indications of thoracocentesis: 1. Any undiagnoesd pleural effusion 2. Therapeutic purposes in massive effusions Collection: 1. Heparinized tubes to avoid clotting 2. Except for an EDTA tube for all counts and differentials Inoculation into the blood culture medium at the bed side * If necessary fresh specimen for cytology may be stored up to 48 hours in the refrigerator with satisfactory results.

Serousal Fluids Pleural Fluid Amylase: measurement of this enzyme is recommended for all pleural effusions with unknown ethiology Increased levels found in esophageal rupture PH value > 7.3 is related to uncomplicated cases PH < 7.2 is related to complicated cases such as bacterial pneumonia, Tb or malignancy PH < 6.0 is characteristic of esophageal rupture Pleural fluid TG > 110 mg/dl indicate a chylous effusion Values between 60-110 mg/dl are less certain and require lipoprotein electrophoresis for chylomicrons Pleural fluid TG < 50 mg/dl indicate a pseudochylous effusion, seen in chronic inflammatory process Adenosine deaminase (ADA) is a rapid chemical evidence of Tb. ADA-2 from lymphocytes

Serousal Fluids Pleural Fluid Formal cell counts have little practical value Pleural fluid Hct > 50% of blood is a good evidence for hemothorax A bloody pleural effusion (Hct >1% or RBC> 100,000/ul) suggest trauma, malignancy and pulmonary infarction Differential cell count on an air-dried Romanowski’s stain Filtration or automated concentration methods with Papanicolaou stain for cytologic evaluation Preparation of cell block is unnecessary except for effusions in which malignancy is a consideration

Serousal Fluids Pleural Fluid Neutrophils: Predaminate in pleural fluid with inflammation. Over 10% of transudates also have a predominance of neutrophils but has no clinical significance Lymphosytes: Associated with transudate and no clinical significance * Most are small but medium, large and reactive variants may be seen * Nuceloi and nuclear cleaving are more prominent in effusions than in prepheral blood * Low grade NHL or CLL may be difficult to distinguish from benign lymphocyte-rich serous effusions. In conjunction with cellular morphology, immunophenotyping by flowcytometry or immunocytochemistry is usually helpful

Serousal Fluids Pleural Fluid Eosinophils: an eosinophilic effusion is defined as having > 10% eosinophils * The most common causes are related to the presence of air or blood in the pleural cavity * Most are exudates * in about 35% of patients the ethiology is unknown * though not of much assistance in diagnosing an effusion, eosinophilia does appear to independently associated with longer survival

Serousal Fluids Pleural Fluid Mesothelial cells: Are common in pleural fluid from inflammatory process. * Rare in patients with Tb pleurisy, empyema, RA and patients who have pleurodesis * Fibrin deposition and fibrosis occurring in these conditions prevent exfoliation of mesothelial cells * Carcinoma cells may form easily recognized tumor clusters or closely mimic mesothelial cells a panel of immunocytochemistry stains may be necessary for conformation

Serousal Fluids Pleural Fluid

Serousal Fluids Pleural Fluid

Serousal Fluids Pleural Fluid

Serousal Fluids Peritoneal Fluid Up to 50 ml Fluid normally present in peritoneal cavity Peritoneal effusion is called Ascites Laboratory criteria for dividing ascitic fluid into transudate and exudate is not well defined as it is for pleural fluid Diagnostic peritoneal lavage (DPL) have limited use: 1. Rapid screening for significant abdominal hemorrhage 2. Evaluation of hollow viscus injuries Peritoneal dialysis: submitted to check for infection Peritoneal washing: performed intra operatively to document early intra abdominal spread of gynecologic and gastric Ca.

Serousal Fluids Peritoneal Fluid Total leukocyte useful in spontaneous bacterial peritonitis (SBP) Approximately 90% of (SBP) have leukocyte count > 500/ul and over 50% neutrophiles Eosinophilia > 10% most commonly associates with chronic peritoneal dialysis. Also in CHF, vasculitis, lymphoma and ruptured hydatid cyst Overall sensitivity of cytology for malignant ascitis is 40-65% Peritoneal carcinomatosis accounts for two thirds of malignant effusions Immunocytochemical stains are useful in characterizing atypical cells

Serousal Fluids Peritoneal Fluid Amylase activity in normal peritoneal fluid is similar to blood levels A fluid amylase level greater than three times of serum value is good evidence of pancreas-related ascitis and also in GI perforation Increased peritoneal BUN and Cr + increased serum BUN + normal serum Cr (due to back diffusion of urea) suggests bladder rupture CEA sensitivity 40-50% specificity 90% using cut off point of 3 ng/ml Increase CEA in peritoneal washing suggest a poor prognosis of gastric Ca CA-125 extremely high in epithelial Ca of ovary, follopian tube or endometrium

Serousal Fluids Peritoneal Fluid

Serousal Fluids Peritoneal Fluid

Serousal Fluids Peritoneal Fluid

Serousal Fluids Pericardial Fluid 10-15 ml fluid normally present in pericardial space Causes of pericardial effusion: 1)infection 2)neoplasm 3)MI 4)hemorrhage 5)methabolic 6)RA HIV infected patients commonly have asymptomatic pericardial effusion In HIV associated cardiac temponade 45% are idiopathic, Tb and bacterial infections each accounts for 20% of cases Large effusions (>350 ml) most often caused by malignancy or uremia Blood-like fluid represent hemorrhagic effusion or aspiration of blood from the heart Hct comparable to peripheral and blood gas analysis help to differentiate

Serousal Fluids Pericardial Fluid Postpericardiotomy syndrome common but nonspecific complication of cardiac surgery, days to weeks following the injury Exudative pericardial effusion developed in over 80% of cases Presence of antimyocardial Abs suggests an immune mediated process Hct and RBC count have limited value in differential diagnosis of pericardial effusions. Total WBC > 10,000/ul suggests bacterial, Tb or malignant pericarditis Metastatic Ca of lung and breast are most frequent cause of malignant pericardial effusion

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