Urinalysis and Body Fluids

CRg

Unit 4
Cerebrospinal Fluid

Cerebrospinal Fluid (CSF)

Produced at the Choroid plexus of the 4 ventricles by modified Ependymal cells
At rate @20 ml / hr (adults) Med training says @ 150 ml/day is produced

CSF flows through the Subarachnoid space

Where a volume of 90 150 ml is maintained (adults)

Reabsorbed at the Arachnoid villus / granulation

to be eventually reabsorbed into the blood

Cerebrospinal Fluid (CSF)

Blood Brain Barrier
Occurs due to tight fitting endothelial cells that prevent filtration of larger molecules. Controls / restricts / filters blood components Restricts entry of large molecules, cells, etc. Therefore CSF composition is unlike bloods ** CSF is NOT an ultrafiltrate

Cerebrospinal Fluid (CSF)

Blood Brain Barrier
Essential to protect the brain Blocks chemicals, harmful substances Antibodies and medications also blocked

Tests for those substances normally blocked can indicate level of disruption by diseases: ie meningitis and multiple sclerosis.

Cerebrospinal Fluid (CSF)

CSF functions
Supplies nutrients to nervous tissues Removes metabolic wastes Protects / cushions against trauma

Cerebrospinal Fluid (CSF)

Four major categories of disease
Meningeal infections Subarachnoid hemorrhage CNS malignancy Demyelinating disease

Cerebrospinal Fluid (CSF)

Indications for analysis
To confirm diagnosis of meningitis Evaluate for intracranial hemorrhage Diagnose malignancies, leukemia Investigate central nervous system disorders

Cerebrospinal Fluid (CSF)

Specimen collection and handling
Routinely collected via lumbar puncture between 3rd & 4th, or 4th & 5th lumbar vertebrae under sterile conditions Intracranial measurement taken before fluid is withdrawn.

Cerebrospinal Fluid (CSF)

Cerebrospinal Fluid (CSF)

Specimen collection and handling
Tube 1 chemistries and serology Tube 2 microbiology cultures Tube 3 hematology

Testing considered STAT Specimen potentially infectious

Cerebrospinal Fluid (CSF)

Specimen collection and handling
If immediate processing not possible
Tube 1 (chem-sero) frozen Tube 2 (micro) room temp Tube 3 (hemo) refrigerated

Cerebrospinal Fluid (CSF)

Appearance
Normal - Crystal clear, colorless Descriptive Terms hazy, cloudy, turbid, milky, bloody, xanthrochromic Often are quantitated slight, moderate, marked, or grossly. Unclear specimens may contain increased lipids, proteins, cells or bacteria. Use precautions.

Cerebrospinal Fluid (CSF)

ASCP 17, CSF tubes over newsprint

Cerebrospinal Fluid (CSF)

Appearance
Xanthrochromic Yellowing discoloration of supernatent (may be pinkish, or orange).
Most commonly due to presence of old blood. Other causes include increased bilirubin, carotene, proteins, melanoma

Cerebrospinal Fluid (CSF)

Appearance

Clots indicates increased fibrinogen & usually due to traumatic tap, but may indicate damage to blood-brain barrier. (see below) Pellicle formation in refrigerated specimen associated with tubercular meningitis. Pellicle formation - picture at right (pellicle in L. tube, R is normal) Milky increased lipids Oily contaminated with x-ray media

Cerebrospinal Fluid (CSF) - procedures

All specimens should be examined microscopically hematology Stat priority, RBC lyse in 1 hour, WBC in 2 hrs. Refrigerate if not able to process immediately. Electronic counters generally unusable. Manual count No dilution usually required (use saline if needed)

ave. # cells counted x dilution # squares counted x volume of each square

Cerebrospinal Fluid (CSF)

Count and differentiate 100 nucleated cells. Any cell found in peripheral blood may be seen in CSF, other nucleated cells and malignant cells can also be found. Entire smear should be evaluated for
abnormal cells, inclusions within cells, Clusters, Presence of intracellular organisms Adults: 70% lymps, 30% monos. Children / newborns: monocyte

Normal differential values

Types of cells
Neutrophils occasionally (with normal count) Macrophages increase following CVA Ependymal cells, and normal lining cells can also be seen.

Cerebrospinal Fluid (CSF)

Lymphocytes & monocytes / macrophages Mono / macro, segs and lymph

L lymphocytes & macrophages

Cerebrospinal Fluid (CSF)

CSF cells
L neutrophiles & @6 macrophages

R neutrophiles and intracellular bacteria

Cerebrospinal Fluid (CSF)

Eosinophils
Often associated with parasitic / fungal infections, allergic reactions including reaction to shunts and other foreign objects.

Cerebrospinal Fluid (CSF)

Ependymal cells
Normal cell, unique to CSF Line the ventricles, produce CSF fluid Large cell with distinct round/oval nucleus, sometimes found in sheets

Cerebrospinal Fluid (CSF)

Suspicious / unclassified or malignant cells are reported as other or unclassified AND are sent to pathology. (as seen below) 1986 CAP CM10 CSF blasts (appearance similar to peripheral blood, always consult with hematology specialist / pathologist) ( see below right)

Cerebrospinal Fluid (CSF)

Cellular inclusions
Erythrophage Siderophage Hematoidin crystals (see below)

Cerebrospinal Fluid (CSF)

CSF Quality Control
Commercial quality control samples available

>>>>>>>>>>>>>>>>>>>>>>>>>>> Chemistry
Blood brain barrier causes selective filtration Abnormal values
from altered permeability Increased production Increased metabolism

Cerebrospinal Fluid (CSF) - protein

Normal 15 45 mg/dL . Albumin fraction. If IgG from damaged B-B, or CNS produced? Can electrophoresis to evaluate oligoclonal / malignant bands. Decreased levels not significant Increases levels Damaged B-B (as in meningitis or hemorrhage) Production of immunoglobulins within CNS (MS) Degeneration of neural tissue Dye-binding methods preferred Alkaline biuret Coomassie brilliant blue - a blue color produced is proportional to the amount of protein present (Beers Law)

Cerebrospinal Fluid (CSF) - glucose

Selectively transported across blood-brain barrier Normal values: 60-70% of blood glucose STAT procedure, glycolysis reduces level quickly. Procedure performed as for blood specimen Decreased levels seen in bacterial & fungal meningitis Hypoglycemia Brain tumors Leukemias Damage to CNS

Differential Diagnosis of Meningitis

by Laboratory Results
Tubercular
Increased WBC count

Bacterial
Increased WBC count

Viral
Increased WBC count

Fungal
Increased WBC count

Neutrophils Marked protein Marked glucose

Lymphs Mod. protein normal glucose

Lymps & Monos Mod-Marked protein glucose

Lymphs & Monos Mod-Marked protein Normal to glucose

Lactate > 35 mg/dL

Lactate normal

Lactate > 25 mg/dL

Lactate > 25 mg/dL

+ gram stains

Pellicle formation

+ India ink with Cryptococcus neoformans

+ immunological test for C. neo.

+ bacterial antigen tests

Cerebrospinal Fluid (CSF)

CSF Lactate
Normal values = 11-22 mg/dL Increase as result of hypoxia Bacterial meningitis. Head injury

CSF Glutamine
Normal 8-18 mg/dL Increased levels associated with increases in ammonia (toxin)

CSF Enzymes
Lactate dehydrogenase (LDH or LD) 5 isoenzyme types; LD1&LD2 are in brain tissue Creatine kinase (CPK or CK) Isoenzyme CK3/ CK-BB from brain tissue Following cardiac arrest, patients with CSF levels <17 mg/dL have favorable outcome.

Cerebrospinal Fluid (CSF)- microbiology

Gram stain Extremely important for early diagnosis of bacterial meningitis Even when well performed, 10% false negatives occur Use of Cytospin to concentrate specimen increases sensitivity Cultures- Aerobic & Anaerobic. Culture blood at same time Organisms Newborns E. coli & group B Strep. Children Streptococcus pneumoniae Hemophilus influenzae Neisseria meningitidis Adults Neisseria meningitidis Streptococcus pneumoniae Staph. aureus (if a shunt is present) Immunocompromised Cryptococcus neoformans, Candida albicans, Coccidioides, or any opportunistic organism

Cerebrospinal Fluid (CSF)

India-ink / nigrosin preparation
Negative stain to view the encapsulated Cryptococcus neoformans (often AIDs /immunocompromised
complication)

Instead of stain, can also use dark field microscopy for same effect. These direct procedures have @ 25-50% sensitivity
Prefer latex agglutination tests, better results

Cerebrospinal Fluid (CSF)

Serology
VDRL (Veneral Disease Research Laboratory)
For detection of neurosyphilis On CSF test low sensitivitiy, but great specificity FTA-Abs also used on CSF, more sensitive, but must prevent blood contamination.

Urinalysis and Body Fluids

CRg

Unit 4
Serous Body Fluids

Serous Fluids
Serous fluids
small amount of fluid that lies between the membranes lining the body cavities (parietal) and those covering the organs within the cavities (visceral).
acts as lubricant, provide nutrients, remove wastes

Serous Fluids
Body cavities
Pericardial - heart Pleural - lungs Peritoneal abdominal

Membranes
Parietal lines cavity wall Visceral covers organs contained within

Serous Fluids
Serous fluid
an ultra filtrate of the plasma closely resembles the plasma
- as opposed to CSF

Production ( parietal membrane) Re-absorption ( visceral membrane)

normally at a constant rate.

Serous Fluids
Production and re-absorption are influenced by:
Changes in osmotic and hydrostatic pressure in the blood Concentration of chemical constituents in the plasma Permeability of blood vessels and the membranes

Serous Fluids
Types of serous fluids
Pericardial fluid around heart Pleural fluid (thoracic fluid) lung cavity Peritoneal (ascitic fluid) abdominal cavity

Reasons for analysis

Infections Hemorrhages malignancies, and other disorders.

Serous Fluids- effusions

Effusion
an increase in the serous fluid due to some disruption in production &/ re-absorption processes. Transudate - an effusion that is a result of a systemic disorder that disrupts the balance of fluid production / fluid reabsorption. (Think that the problem has been transferred. Exudates term to classify the effusion that is a result of a problem with the membranes themselves.
Thought of as an inflammatory process Produced by conditions that directly involve the membranes of the particular cavity, ex. infections, inflammation, and malignancies o Pleural exudate carcinoma, pneumonia, trauma o Pericardial exudate infection, cardiovascular disease (CV) trauma, cancer

Examples: Pleural transudate congestive heart failure; Pericardial transudate nephrotic syndrome, metastatic cancer

Serous Fluids
Specimen Collection and Handling
Needle aspiration
Paracentesis Thoracentesis Pericardiocentesis

Lavage ( ie. peritoneal lavage)

Ringers lactate / saline is infused into abdomen then retrieved for analysis. Specimen sometimes called ascites fluid.

Serous Fluids
Specimen Collection and Handling
EDTA tube for cell count & differential Heparin tube for chemistries, serology, microbiology and cytology. Since procedure not performed unless an effusion exists, large amount of fluid often collected. Blood specimens usually collected at same time and comparisons of test results made.

Serous Fluids
Variety of tests used to aid in determining the cause of the effusion: appearance, protein level, LDH level, cell counts, whether or not it will form a clot, etc. Authors recommend fluid to blood ratios of protein and LDH. Protein in fluid / protein in serum LDH in fluid / LDH in serum

General lab procedures for serous fluids

Hematology
Color & clarity NV = yellow & clear (other terms as for CSF are sometimes used, EXCEPT xanthrochromic Cell count same as for CSF Differential
any cell in peripheral blood, mesothelial cells, (cells lining these cavities more info soon) malignant cells

General lab procedures for serous fluids

Chemistry (varies depending on which fluid)

Total protein & enzymes (LDH and ratio to serum LDH) Glucose Amylase & Lipase pancreatic disorders Bilirubin - peritoneal fluid suspicion of perforated GI or gall bladder Alkaline phosphatase - peritoneal fluid suspicion of perforated intestine pH & ammonia

Serous Fluids
Microbiology
Gram stain & acid fast Cultures aerobic & anaerobic

Serous Fluids
Serology
rarely done. Serum results better.

Cytology
if malignancy suspected

Quality control no commercial controls, must use serum controls.

Serous Fluids
Mesothelial cells
Unique to serous fluids, originate from lining of peritoneal, pleural, and pericardial cavities. Large round cell with abundant blue cytoplasm and purple nucleus which may be eccentric Cell sometimes described as having a "fried egg" appearance. - usually are single or may

be in sheets Nucleus round to oval & has a smooth outline, takes up @ 1/3 - of the space. Smooth spherical nuceoi may be seen.

Serous Fluids
Mesothelial cells
Pleomorphic If reactive may appear in clusters, have prominent nucleoli and be multinucleated Nucleus still distinct and round with uniform staining characteristics. A cluster of reactive mesos may resemble malignant cell clusters, but the mesos display "cell windows."

Serous fluid - Mesothelial cell

Mesothelial cell

Serous Fluids
lymphocyte, macrophage/mono, basophil Mesothelial cells

Serous Fluids
Macrophage engulfed Candidia species in a pleural fluid, mesothelial cells.

Serous Fluids
1987CAP CM-16 Pleural fluid mesothelial cell

Serous Fluids
1992 cap CM-20 Pleural fluid, seg, lymph, macrophage,

Serous Fluids
Pleural fluid mesothelial cells

Serous Fluids
ASCP 3 reactive mesothelial cells

Serous Fluids
Malignant cells
A frequent concern in any serous fluid due to possibility of cancer of any organ and/or metastasis of CA from one location to another. Cells have irregular size, shape, and staining characteristics of nucleus and cytoplasm. Usually deeply basophilic, molded or balled up clusters of cells with little distinction from one cell to the next. May be vacuolated.

Serous Fluids
Malignant cells
Characteristics
Irregular shape Uneven chromatin distribution Prominent large irregular nucleoli, Community borders Increased nuclei / cytoplasm ratio.

Always send suspicious cells to cytology / pathology

Serous Fluids
Malignant cells ACSP 7 Case 1 cont. peritoneal fluid, malignancy

Serous Fluids
Malignant cells
ASCP 9 Case 2 pleural fluid 42 year old, breast cancer

Serous Fluids
Malignant cells
ASCP 10 Case 3, ascitic fluid, 62 year old admitted for GI bleeding

Serous Fluids
Malignant cells
ASCP 11 Case 3, ascitic fluid, 62 year old admitted for GI bleeding. Liver biopsy, H&E stain. Nest of malignant cells.

Serous Fluids
Malignant cells
ASCP 12 Case 4, 30 year old with back pain and inability to work. Pleural effusion fluid malignant tumor on spinal cord

Serous Fluids
Malignant cells
ASCP 12 Case 4, 30 year old with back pain and inability to work. Pleural effusion fluid malignant tumor on spinal cord

Serous Fluids
Malignant cells
1992 CAP CM-31 peritoneal fluid malignant cell

Serous Fluids
Malignant cells
sheets of atypical cells with irregular nuclear contours, nuclear hyperchromasia, basophilic cytoplasm, and jagged outline of cell borders. Squamous cell carcinoma (x400 , Diff-Quik staining)

Serous Fluids-LE cells

Seen in patients with Systemic Lupus Erythmatosis (SLE) a systemic disease in which an autoantibody attacks the patients organs and body systems LE cell is a neutrophil that has engulfed a homogeneous mass of purple staining nuclear material

Serous Fluids pericardial fluid

1987 CAP CM21 Pericardial fluid, intracellular bacteria

Serous Fluids peritoneal fluid

1992 CAP CM41 Peritoneal fluid. Seg, macro, yeast

Serous Fluids peritoneal fluid

1992 CAP CM40 Peritoneal fluid. Seg, macro, yeast

Abdominal fluid plasma cells / multiple myeloma

Serous Fluids
1991 CAP CM 20