Pathological examination of body fluids

examination of CSF, Pleural fluid

1. EXAMINATION OF BODY FLUIDS Dr Utkarsh Sharma Moderated by: Dr Navita Gupta

2. EXAMINATION OF CSF In adults, approximately 500 mL of cerebrospinal fluid (CSF) is produced each day (0.3 to 0.4 mL/min). The total adult volume varies from 90 to 150 mL. In neonates, the volume varies from 10 to 60 mL . the total CSF volume is replaced every 5 to 7 hours

3. The normal opening adult pressure is 90 to 180 mm of water in the lateral decubitus position with the legs and neck in a neutral position . Opening pressures above 250 mm H2O are diagnostic of intracranial hypertension. If the opening pressure is greater than 200 mm H2O in a relaxed patient, no more than 2.0 mL should be withdrawn.

4. The CSF specimen is usually divided into three serially collected sterile tubes: Tube 1 for chemistry and immunology studies; T ube 2 for microbiological examination T ube 3 for cell count and differential . An additional tube may be inserted in the No. 3 position for cytology if a malignancy is suspected . Note: Tube 1 should never be used for microbiology because it may be contaminated with skin bacteria.

5. Glass tubes should be avoided because cell adhesion to glass affects the cell count and differential . Specimens should be delivered to the laboratory and processed quickly to minimize cellular degradation, which begins within 1 hour of collection . Refrigeration is contraindicated for samples in which flow cytometry is likely to be needed for detection of leukemia or lymphoma cells.

6. GROSS EXAMINATION Normal CSF is crystal clear and colorless and has a viscosity similar to that of water. Clot formation may be present in patients with traumatic taps, complete spinal block ( Froin’s syndrome), or suppurative or tuberculous meningitis. It is not usually seen in patients with subarachnoid hemorrhage. Clots may interfere with cell count accuracy by entrapping inflammatory cells and/or by interfering with automated instrument counting

7. Viscous CSF may be encountered in patients with Metastatic mucin producing adenocarcinomas. Cryptococcal meningitis due to capsular polysaccharide L iquid nucleus pulposus resulting from needle injury to the annulus fibrosus .

8. Pink-red CSF usually indicates the presence of blood and is grossly bloody when the RBC count exceeds 6000/ μL . It may originate from a subarachnoid hemorrhage, intracerebral hemorrhage, or cerebral infarct or from a traumatic spinal tap . Xanthochromia commonly refers to a pale pink to yellow color in the supernatant of centrifuged CSF.

9. Visible CSF xanthochromia may also be due to the following: ( 1) oxyhemoglobin resulting from artifactual red blood cell lysis caused by detergent contamination of the needle or collecting tube, or a delay of longer than 1 hour without refrigeration before examination; ( 2) bilirubin ( bilirhachia ) in jaundiced patients ; ( 3) CSF protein levels over 150 mg/ dL , ( 4) disinfectant contamination; ( 5) carotenoids (orange) in people with dietary hypercarotenemia (i.e., hypervitaminosis A ); ( 6) melanin (brownish ) from meningeal metastatic melanoma; and ( 7) rifampin therapy (red-orange ).

10. Differential Diagnosis of Bloody CSF A traumatic tap occurs in about 20% of lumbar punctures. Distinction of a traumatic puncture from a pathologic hemorrhage is, therefore, of vital importance. In a traumatic tap, the hemorrhagic fluid usually clears between the first and third collected tubes but remains relatively uniform in subarachnoid hemorrhage. Xanthochromia , microscopic evidence of erythrophagocytosis , or hemosiderin-laden macrophages indicate a subarachnoid bleed in the absence of a prior traumatic tap. RBC lysis begins as early as 1 to 2 hours after a traumatic tap. Thus, rapid evaluation is necessary to avoid false-positive results.

11. MICROSCOPIC EXAMINATION Total Cell Count The normal leukocyte cell count in adults is 0 to 5 cells/ μL . It is higher in neonates, ranging from 0 to 30 cells/ μL , with the upper limit of normal decreasing to adult values by adolescence . No RBCs should be present in normal CSF. If numerous (except with a traumatic tap), a pathologic process is probable (e.g., trauma, malignancy, infarct, hemorrhage).

12. Differential Cell Count A differential performed in a counting chamber is unsatisfactory because the low cell numbers give rise to poor precision, and identifying the cell type beyond granulocytes and “ mononuclears ” is difficult in a wet preparation. Direct smears of the centrifuged CSF sediment are also subject to significant error from cellular distortion and fragmentation . The cytocentrifuge method is rapid, requires minimal training, and allows Wright’s staining of air-dried cytospins . It is the recommended method for differential cell counts in all body fluids.

13. In adults, normal CSF contains small numbers of lymphocytes and monocytes in an approximate 70 : 30 ratio. A higher proportion of monocytes is present in young children, in whom up to 80% may be normal. No general consensus regarding an upper limit of normal for PMNs has been established . Many laboratories accept up to 7% neutrophils with a normal WBC count . The number of PMNs may be decreased by as much as 68% within the first 2 hours after lumbar puncture owing to cell lysis . A total PMN count of over 1180 cells/ μL (or more than 2000 WBCs/ μL ) has a 99% predictive value for bacterial meningitis.

14. Cerebrospinal fluid cytology (lymphocyte to monocyte distribution ratio 70 : 30).

15. Choroid plexus cells in cerebrospinal fluid.

16. Cluster of blastlike cells in cerebrospinal fluid from a premature newborn.

17. Hemosiderin-laden macrophages ( siderophages ) from the cerebrospinal fluid of a patient with subarachnoid hemorrhage. Hemosiderin crystals (golden-yellow) are also present.

18. Acute lymphoblastic leukemia in cerebrospinal fluid. Note uniformity of the blast cells.

19. Burkitt’s lymphoma in cerebrospinal fluid. The cells are characterized by blue cytoplasm with vacuoles and a slightly clumped chromatin pattern.

20. CSF protein levels of 15 to 45 mg/ dL have long been accepted as the “normal” reference range.

21. EXAMINATION OF PLEURAL FLUID The pleural cavity is a potential space lined by mesothelium of the visceral and parietal pleurae . The pleural cavity normally contains a small amount of fluid that facilitates movement of the two membranes against each other . An accumulation of fluid, called an effusion, results from an imbalance of fluid production and reabsorption . Except for an EDTA tube for total and differential cell counts, the specimen should be collected in heparinized tubes to avoid clotting.

22. Because serous effusions are more forgiving than CSF in maintaining cellular integrity, fresh specimens for cytology may be stored for up to 48 hours in the refrigerator with satisfactory results I nitial classification of a pleural fluid as a transudate or an exudate greatly simplifies the process of arriving at a correct final diagnosis . Transudates generally require no further workup.

23. GROSS EXAMINATION Transudates are typically clear, pale yellow to straw-colored, and odorless, and do not clot. Approximately 15% of transudates are blood tinged. A bloody pleural effusion (hematocrit >1%) suggests trauma, malignancy, or pulmonary infarction . A traumatic tap is suggested by uneven blood distribution, fluid clearing with continued aspiration, or formation of small blood clots. A pleural fluid hematocrit greater than 50% of the blood hematocrit is good evidence for a hemothorax .

24. Exudates may grossly resemble transudates, but most show variable degrees of cloudiness or turbidity, and they often clot if not heparinized. Turbid , milky or bloody specimens should be centrifuged and the supernatant examined. If the supernatant is clear, the turbidity is most likely due to cellular elements or debris. If the turbidity persists after centrifugation, a chylous or pseudochylous effusion is likely.

25. Cell Counts Leukocyte counts have limited utility in separating transudates (< 1000/ μL ) from exudates (>1000/ μL ). Although RBC counts above 100,000/ μL are highly suggestive of malignancy, trauma, or pulmonary infarction , they are not specific for these conditions . Mesothelial cells are common in pleural fluids from inflammatory processes, however, conspicuously scarce in patients with tuberculous pleurisy and rheumatoid pleuritis , and in patients who have had pleurodesis . Nucleoli and nuclear cleaving are more prominent in lymphocytes of effusions than in the peripheral blood.

26. Mesothelial cells in pleural fluid

27. Small cell carcinoma of the lung showing typical molding of nuclei .

28. EXAMINATION OF PERITONEAL FLUID Ascites is the pathologic accumulation of excess fluid in the peritoneal cavity . Up to 50 mL of fluid is normally present in this mesothelial -lined space. Diagnostic paracentesis is performed in most patients with new ascites, or if there is a change in the clinical picture of a patient with ascites, such as rapid fluid accumulation or fever development. A minimum of 30 mL is needed for complete evaluation . If possible, at least 100 mL should be provided for cytologic examination.

29. GROSS EXAMINATION T ransudates are generally pale yellow and clear. Exudates are cloudy or turbid because of the presence of leukocytes, tumor cells, or increased protein levels. The presence of food particles, foreign material, or green-yellow bile staining in a DPL specimen suggests perforation of the gastrointestinal or biliary tract . Acute pancreatitis and cholecystitis may also cause greenish discoloration. As little as 15 mL of blood per liter of fluid produces a bright red opaque color. Bloody ascites is also seen in malignancy and tuberculosis.

30. MICROSCOPIC EXAMINATION The total leukocyte count is useful in distinguishing ascites due to uncomplicated cirrhosis from spontaneous bacterial peritonitis (SBP), which is caused by migration of bacteria from the intestine into the ascitic fluid. Approximately 90% of patients with SBP will have leukocyte counts greater than 500/ μL , more than 50% of which are neutrophils. Eosinophilia (>10%) is most commonly associated with the chronic inflammatory process associated with chronic peritoneal dialysis, lymphoma , and ruptured hydatid cyst.

31. Semen Analysis In addition to its use in the evaluation of reproductive dysfunction, particularly infertility , semen analysis is used to select donors for therapeutic insemination and to monitor the success of surgical procedures, such as varicocelectomy and vasectomy. The patient should be instructed to collect semen after 2 to 5 days of sexual abstinence. Longer periods of abstinence usually result in a higher semen volume but reduced sperm motility. In such a case, a second semen specimen may be collected 2 hours after the first sample is collected.

32. The semen specimen should be delivered to the laboratory within 1 hour of collection and kept warm during transportation . Two specimens collected within 2- to 3-week intervals should be used for evaluation, and if they are markedly different, additional specimens should be collected . Semen should be thoroughly mixed before examination, and its viscosity recorded . The appearance of a yellow hue in a semen specimen is associated with pyospermia , and a rust color is due to small bleedings in the seminal vesicle.

33. A hemocytometer or microchamber should be used for the sperm count . Total sperm count is then calculated by multiplying the dilution factor (normal concentration range, 15-50 million/mL) by its volume (normal range, 2-5 mL ). Progressive motility (normal range 32% or above) is expressed as the percentage of sperm that move. In addition, forward movement is graded. Sperm that move rapidly in a straight line with little yaw and lateral movement are grade 4 if they move more slowly, grade 3. Grade 2 sperm move even more slowly and with substantial yaw. Grade 1 sperm have no forward progression . Zero progression denotes absence of any motility .

34. If motility is less than 50%, a viability stain of eosin Y with nigrosin as a counterstain is done. In bright-field microscopy, dead sperm will stain red, whereas live sperm will exclude the dye and appear unstained . In samples with no visible sperm, such as postvasectomy semen , the entire sample should be centrifuged and the pellet examined for intact or damaged sperm fragments. The analysis should be repeated in 4 to 6 months.

35. Agglutination occurs when motile sperm stick to one another in an orientation that is reproducible within a given specimen, such as head to head , tail to tail, midpiece to midpiece , or mixed ways, depending on the specificity of sperm antibodies. Agglutination suggests an immunologic cause of infertility, and a description of the type of agglutination should be recorded. This can usually be distinguished from clumping due to bacterial infection or tissue debris, which typically involves nonspecific orientation of the sperm.

36. Round cells should be differentiated into two classes: immature germ cells with a single or double highly condensed nucleus with a relatively large area of cytoplasm, and polymorphonuclear leukocytes, which are smaller than the germ cells and have a lower nuclear/cytoplasmic ratio. Peroxidase staining specifically identifies the polymorphonuclear leukocytes in the presence of lymphocytes and other cells that normally occur in semen . Bacterial contamination should be noted, as should the presence or absence of epithelial cells.

37. Typically, more than 4% of the sperm in a semen specimen should exhibit normal morphology . A strict morphology score of greater than 4% of normal indicates excellent fertilizing capacity . Scores between 0% and 3% predict probable inability to fertilize . Wide variability in the size of the acrosomal cap is the most obvious characteristic of abnormal sperm. An acrosomal cap less than one third of the head surface is considered abnormal, as are retention of a cytoplasmic droplet greater than one-half of the head size and a tail less than 45 μm long. Of particular note is the direct relationship between acrosome size and the frequency of fertilization or pregnancy.