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

2. 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

3. 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

4. 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

52. Thank You

53. 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 : ml in adults and ml in neonates
In adults up to 20 ml CSF can be removed

54. 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

55. 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

56. CSF
Xanthochromia: pink, orange or yellow CSF due to RBC lysis and Hb breakdown
Peak density occurs at about 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

57. 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 RBC

58. 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

59. 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 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%)

60. CSF Over 80% of CSF protein is derived from plasma (1% of blood level)
Protein concentration increases caudally, Mean lumbar value ranges between 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 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

61. CSF

62. CSF

63. CSF

64. CSF

65. CSF

67. 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)

68. 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

69. Serousal Fluids Transudate Exudate Appearance Clear Cloudy
Specific gravity < > 1.015
Total protein < 3.0 gr/dl > 3.0 gr/dl
F/S protein ratio < > 0.5
LD < 200 IU > 200 IU
F/S LD ratio < > 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

70. 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

71. 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.

72. 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 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

73. 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

74. 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

75. 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

76. 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

77. Serousal Fluids
Pleural Fluid

78. Serousal Fluids
Pleural Fluid

79. Serousal Fluids
Pleural Fluid

80. 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.

81. 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

82. 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

83. Serousal Fluids
Peritoneal Fluid

84. Serousal Fluids
Peritoneal Fluid

85. Serousal Fluids
Peritoneal Fluid

86. 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

87. 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

88. Thank You