BODY FLUID ANALYSIS FOR CELLULAR COMPOSITION Afsaneh Rajabiani, APCP Tehran Medical University...
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Transcript of BODY FLUID ANALYSIS FOR CELLULAR COMPOSITION Afsaneh Rajabiani, APCP Tehran Medical University...
BODY FLUID BODY FLUID ANALYSIS FOR ANALYSIS FOR
CELLULAR CELLULAR COMPOSITIONCOMPOSITION
Afsaneh Rajabiani, APCPAfsaneh Rajabiani, APCP
Tehran Medical UniversityTehran Medical University
Shariati Hospital Shariati Hospital
Morphology Assessment Morphology Assessment of Body Fluidof Body Fluid
Wedge smears (push smears) should Wedge smears (push smears) should notnot be used with fluids be used with fluids because of their inferior ability in preserving intact cells.because of their inferior ability in preserving intact cells.
The cytocentrifuge preparation is recommended for air-dried body The cytocentrifuge preparation is recommended for air-dried body fluid slides, because:fluid slides, because:
Concentrate the cells Concentrate the cells Minimizes cell distortionMinimizes cell distortion Produces a monolayer of cellsProduces a monolayer of cells Romanowsky-type stained slides show excellent morphologic Romanowsky-type stained slides show excellent morphologic
detaildetail Cell typically are randomly dispersed in a small circular areaCell typically are randomly dispersed in a small circular area When malignancy is suspected, the whole cellular area should be When malignancy is suspected, the whole cellular area should be
evaluated evaluated
Morpholoy Assessment of Morpholoy Assessment of Body FluidBody Fluid
Cells are concentrated approximately 20-fold by Cells are concentrated approximately 20-fold by cytocentrifugationcytocentrifugation
*Even hypocellular samples with a chamber cell *Even hypocellular samples with a chamber cell count of zero can have a yield of approximately count of zero can have a yield of approximately 35 cells per slide.35 cells per slide.
The quantitative yield, however, varies from 30 The quantitative yield, however, varies from 30 to 75%, and smaller cells, such as lymphocytes, to 75%, and smaller cells, such as lymphocytes, may be underrepresentatedmay be underrepresentated
The speed and time of centrifugation, the The speed and time of centrifugation, the amount of sample in the chamber, and the filter amount of sample in the chamber, and the filter paper absorbance are factors that can influence paper absorbance are factors that can influence both the cell yield and morophologyboth the cell yield and morophology
Morphology Assessment Morphology Assessment of Body Fluidof Body Fluid
Fresh, unfixed specimens should be used, particularly Fresh, unfixed specimens should be used, particularly in body fluid samples with low protein contents (such in body fluid samples with low protein contents (such as CSF)as CSF)
Prolonged delay in preparing slides (more than 8 Prolonged delay in preparing slides (more than 8 hours), the report should include a statement that the hours), the report should include a statement that the differential count may be inaccurate, due to cellular differential count may be inaccurate, due to cellular degenerationdegeneration
Body fluids may contain fibrin and other proteins that Body fluids may contain fibrin and other proteins that can clog the filter cardcan clog the filter card
Washing the cells by centrifuging the sample and Washing the cells by centrifuging the sample and resuspending in saline, improve the cell yeild and resuspending in saline, improve the cell yeild and morphology morphology
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
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
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
CSF
CSF
CSF
CSF
CSF
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
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
Transudate and Exudate
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
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
Pleural Fluid
Serousal Fluids
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.
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 FluidsPleural Fluid
Serousal Fluids
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
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 FluidsPleural 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 FluidsPleural 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 FluidsPleural Fluid
Serousal FluidsPleural Fluid
Serousal FluidsPleural Fluid
Serousal FluidsPleural 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 FluidsPeritoneal 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 FluidsPeritoneal 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 FluidsPeritoneal Fluid
Serousal FluidsPeritoneal Fluid
Serousal FluidsPeritoneal Fluid
Serousal FluidsPeritoneal 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 FluidsPericardial 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
Serousal FluidsPericardial Fluid