Bio 125 Cerebrospinal Fluid Lecture 2

Malignant cells can have some (but not all) of the following morphological characteristics:

(1) non-uniform cell size (2) N:C ratio of 4:1 to 6:1 (3) variation in size, shape, number and appearance of nuclei in each cell (4) prominent nucleoli (5) crowding or moulding of the cells (6) cytoplasm that is well or irregularly defined (7) varying amounts of cytoplasmic vacuolation, and (8) rosette formation (ball-like) in some types of cancer

State the normal concentration of total protein in the CSF.

Chemistry Tests: Total Protein Normal: 15 - 45 mg/dL

State the normal values of glutamine in CSF and the significance of abnormal concentrations.

Glutamine Normal: 8 - 18 mg/dL Abnormal: > 35 mg/dL associated with disturbance of consciousness (toxicity of ammonia to CNS)

Explain the reason for performing lactate tests on CSF.

High: tubercular meningitis or fungal meningitis Higher: bacterial meningitis and to rule out viral meningitis

Increased numbers of neutrophils can be found in the CSF of patients with

bacterial, early viral, tuberculous, and fungal meningitis, cerebral abscess, subarachnoid and intracerebral hemorrhage, and tumors.

List 3 functions of CSF.

-Protects brain and spinal cord from sudden changes in pressure -Maintains a stable chemical environment -Removes waste products

Do lymphocyte have a nucleus?

-all white blood cells do, that stains dark blue to purple with a Wright's stain. -occupies at least one-half to two-thirds the area of the cell. -2:1 to 4:1 depending on the lymphocyte.

State 6 reasons why a physician requests a laboratory examination of CSF.

1&2. Obtain for diagnosis of disease like Infections or Tumors 3. Measure and relieve cerebrospinal pressure 4. Administer anticancer drugs 5. Detect spinal subarachnoid block 6. Administer antibiotics

State the normal values for red blood cells and white blood cells in CSF.

1. 0-10 RBC/microliter 2. 0 - 5 WBC/uL mostly lymphocytes and monocytes

Discuss the formation, circulation and reabsorption of CSF within the CNS.

1. CSF is formed from the choroid plexus associated with ventricles (spaces) in the brain. 2. The fluid exits from the ventricles through three openings near the base of the brain into the subarachnoid space and through another opening into the inside the spinal cord. 3. CSF circulates in the subarachnoid space around the brain and spinal cord. 4. CSF is returned to the superior sagittal sinus (blood vessel) through the arachnoid villus (villi, pleural).

Discuss the rationate for collecting CSF in 3 separate tubes and name the location in the laboratory where each tube is examined.

1. Chemistry/Serology (Total protein, albumin, IgG, Protein electrophoresis, Myelin-basic protein, Glucose, Lactate, Glutamine, LDH isoenzymes) 2. Microbiology (gram stain, culture, India ink preparation) 3. Hematology (RBC count, WBC count, WBC differential and morphology evaluation)

Describe the location of the 3 layers of meninges within the central nervous system (CNS).

1. Dura mater - tough, outer layer 2. Arachnoid - thin and delicate; lies very close to dura mater 3. Pia mater - lies directly on brain and spinal cord; innermost layer

Monocytes,representing what % WBC

2-6% of all circulating white cells in normal blood, are the largest in size. Each monocyte is about 2-3 times the size of a red blood cell.

CASE STUDY 2 A 2-year-old boy suddenly became ill at a day-care center and his parents were notified. By the time his parents got to the day-care center, the child had a fever of 1030 and was vomiting. He told his parents that his head "hurt" and that he couldn't move his neck. He was also very drowsy. His parents immediately took him to the emergency room at a local hospital. He was examined by a physician and a lumbar puncture was performed. Three tubes of CSF were examined by the laboratory. The results of those examinations were: Chemistry examination: protein -- moderately elevated glucose -- 55 mg/dL (55 mg%) lactate -- 11 mg/dL Microbiology examination: gram stain -- negative culture -- negative India ink preparation -- negative Hematology examination: RBC count -- 8 RBC/uL WBC count -- 300 WBC/uL WBC differential and morphology evaluation: neutrophils -- 5% monocytes -- 5% lymphocytes -- 90% It was noted on the slide that there was a great variation in the size and morphological features of the lymphocytes. Several "reactive" lymphocytes were also reported similar to the one identified at the tip of the pointer.

Answer the following questions based on your analysis of the above information: 1. What is the most likely diagnosis for this child's disease? 2. Identify the abnormal symptoms described above. 3. Which of the laboratory tests are abnormal? 4. Which of the LDH isoenzymes do you expect to be elevated? 5. Why isn't the India ink preparation positive?

CASE STUDY 1 An 82-year-old patient was admitted to the hospital complaining of a headache, vomiting, fever, and convulsions. A lumbar puncture was performed by a physician in the emergency room with no trauma. Three tubes of CSF were collected and one each taken to the chemistry, microbiology and hematology departments of the laboratory for evaluation. It was noted that all three tubes were the same "red" color. Several tests were performed on the samples in each of the three departments. The results included the following: Chemistry examination: protein -- 185 mg/dL glucose -- 85 mg/dL lactate -- 15 mg/dL (15 mg%) LDH isoenzymes -- LD1 and LD2 are elevated Microbiology examination: gram stain -- no organisms seen culture -- no growth India ink preparation -- negative Hematology examination: RBC count -- 1,552 RBC/uL WBC count -- 410 WBC/uL WBC differential and morphology evaluation: neutrophils -- 59% lymphocytes -- 36% monocytes -- 4% eosinophils -- 1% It was also noted that there were many macrophages seen on the slide that contained ingested RBC. When a special stain for iron was performed, large amounts of iron were demonstrated within the cytoplasm of the macrophages.

Answer the following questions based on your analysis of the above information: 1. What is the most likely diagnosis for this patient? 2. Which patient symptoms are abnormal? 3. What laboratory tests are abnormal? 4. Explain the significance of the "red" color of the CSF. 5. Why are LD1 and LD2 isoenzymes elevated?

Plasma cells are derived from

B lymphocytes in the bone marrow.

Differentiate between the CSF findings in bacterial, viral, tubercular and fungal meningitis.

Bacterial Meningitis: 1. Elevated WBC count 2. Neutrophils present 3. Marked protein elevation 4. Markedly decreased glucose 5. Elevated lactate 6. Elevated LD4 and LD5 7. Positive gram stain, bacterial antigen Viral Meningitis: 1. Elevated WBC count 2. Lymphocytes present 3. Moderate protein elevation 4. Normal glucose 5. Normal lactate 6. Elevated LD2 and LD3 Tubercular Meningitis: 1. Elevated WBC count 2. Lymphocytes and monocytes present 3. Moderate to marked protein elevation 4. Decreased glucose 5. Elevated lactate 6. Pellicle formation Fungal Meningitis: 1. Elevated WBC count 2. Lymphocytes and monocytes present 3. Moderate to marked protein elevation 4. Normal to decreased glucose 5. Elevated lactate 6. Positive India ink stain with 7. Cryptococcus neoformans 8. Positive latex test

Nucleated red blood cells (NRBC) are not normally seen in

CSF

Ependymal cells are not normally found in

CSF but sometimes they are dislodged during specimen collection or other surgical procedures and may appear in a CSF specimen. They are usually seen in sheets of cells and are not clinically significant.

Explain the procedure for collecting CSF including the important anatomy at the site of collection.

CSF is routinely collected from the subarachnoid space at the L3-L4 levels of the vertebral column below the termination of the spinal cord at vertebral column level L2.

Explain the cause(s) for the following abnormal CSF: Pellicle

Cause/Major Clinical Significance 1. Protein/Disorders that affect blood-brain barrier 2. Clotting factors/Tubercular meningitis

Explain the cause(s) for the following abnormal CSF: Clotted

Cause/Major Clinical Significance 1. Protein/Disorders that affect blood-brain barrier and Production of IgG within CNS 2. Clotting factors fibrinogen /Traumatic tap

Explain the cause(s) for the following abnormal CSF colors: red

Cause/Major Clinical Significance= red 1. RBC/Hemorrhage 2. Oily/Radiographic contrast media

Explain the cause(s) for the following abnormal CSF colors: xanthochromic (yellow to brown) mostly pink, orange, or yellow

Cause/Major Clinical Significance= xanthochromic mostly RBC degredation 1. Hemoglobin/Old hemorrhage, Lysed cells from traumatic tap 2. Bilirubin/RBC breakdown, Elevated serum bilirubin 3. Merthiolate/Contamination 4. Carotene/Increased serum levels 5. Protein/Disorders that affect blood-brain barrier, Production of IgG within CNS 6. Melanin/Meningeal melanosarcoma

Explain the cause(s) for the following abnormal CSF colors: cloudy, turbid, milky

Cause/Major Clinical Significance=cloudy, turbid, milky 1. WBCs/Meningitis 2. RBCs/Hemorrhage; traumatic tap 3. Microorganisms/Meningitis 4. Protein/Disorders that affect blood-brain barrier Production of IgG within CNS

Explain the reason for performing glutamine tests on CSF.

Glutamine Indirectly reflects CNS ammonia levels. Conversion of ammonia to glutamine protects CNS

Name 3 additional tests that could be performed during a CSF evaluation.

Immunology: Tests for bacterial and fungal antigens Latex agglutination Enzyme-linked immunosorbent assay (ELISA) Serology: Tests for syphilis VDRL (Venereal Disease Research Laboratories) FTA-ABS (Fluorescent treponemal antibody-absorption) Bacterial and viral Meningitis:

State the normal values of lactate in CSF and the significance of abnormal concentrations.

Lactic Acid (Lactate) Normal: 10 - 20 mg/dL Increased above 35 mg/dL: bacterial meningitis and to rule out viral meningitis Increased above 25 mg/dL: tubercular meningitis or fungal meningitis

Differentiate between the types of LDH isoenzymes found in brain tissue, lymphocytes and neutrophils.

Lactic dehydrogenase: 1. Isoenzymes LD1 and LD2 are in brain tissue 2. Isoenzymes LD2 and LD3 are in lymphocytes 3. Isoenzymes LD4 and LD5 are in neutrophils

Are macrophages counted in WBC differential?

Macrophages are not normally in circulation and are not counted in a WBC differential.

Discuss the significance of each of the 3 types of microbiological stains and preps that are used to examine CSF.

Microbiology: Gram stain for bacteria meningitis: 1. Acid-fast stain for tubercular meningitis 2. India ink preparation for crytococcal meningitis (Cryptococcus neoformans) 3. Culture

Define myelin basic protein and discuss its clinical significance.

Myelin Basic Protein: One of the proteins in myelin (proteins and lipids) Released in demyelinating diseases such as multiple sclerosis

Compare the normal concentration of glucose in CSF with the concentration of glucose in blood.

Normal: 50 - 80 mg/dL 60 - 70% of plasma concentration Decreased: bacterial meningitis or tubercular meningitis

a normal small lymphocyte and a cell which is sometimes called

a "plasmacytoid" lymphocyte because its morphology shows some characteristic features of a true plasma cell. The plasmacytoid lymphocyte at the tip of the pointer is making antibodies.

Adjacent to the nucleus of plasma cell is

a lighter stained area ("hof") which is location of the Golgi apparatus within the cell.

The nucleus in a monocyte stains

a more reddish tint. not dark blue as it does in a lymphocyte. This is because the nuclear material in a monocyte is not as dense as the nuclear material in a lymphocyte or neutrophil.

cytoplasm of neutrophils stains

a pinkish-gray color and is loaded with numerous small purple granules.

histiocytes are another name for

another name for macrophages

Malignant cells

appears to be nucleus surrounded by a thin rim of cytoplasm. The cytoplasm is also very irregular on its edges. Crowding of cells is also apparent in this specimen.

Macrophages are very large cells which are derived from

blood monocytes that have migrated into the tissues.

NRBC are found in the

bone marrow as precursors of mature, non-nucleated RBC.

During a traumatic tap, the aspiration needle may actually penetrate the bone and some of these early precursor cells

can be drawn into the needle and appear in the CSF sample.

State the normal appearance of CSF.

clear and colorless

Describe the differences in appearance between a CSF sample from a patient with a cerebral hemorrhage and a CSF sample collected during a traumatic spinal tap.

cloudy, turbid, milky: RBCs/Hemorrhage; traumatic tap red: RBCs/Hemorrhage xanthochromic: Hemoglobin/Old hemorrhage

Monocyte nucleus are shaped like what

contains a nucleus that is horseshoe-shaped, kidney-shaped or indented.

Choroid plexus cells are specialized

cuboidal cells that project into the ventricles of the brain and produce cerebrospinal fluid by ultrafiiltration of the blood plasma.

Hemosiderin in the cytoplasm stains

deep purple and is the iron-containing pigment that is formed when hemoglobin is broken down.

plasma cells can often be found in CSF.

during certain disease states such as tuberculous, syphillis and multiple sclerosis,

Plasma cells contain an _______ nucleus

eccentrically-placed

granules in the cytoplasm contain

enzymes that aid in the killing and digestion of bacteria and fungi.

Ependymal cells are

epithelial cells that form the lining of the ventricles of the brain and the central canal of the spinal cord.

(T-cells) participate

in the cell-mediated immune response

Plasma cells are normally not found

in the circulating blood or CSF.

(B-cells) participate

in the humoral immune response.

Grossly bloody CSF can be an indication of

intracranial hemorrhage, but is may al be from a blood vessel puncture during a spinal tap. Blood from a cerebral hemorrhage will be evenly distributed throughout the three CSF specimen tubes, where as a traumatic tap will leave the heaviest concentration of blood in tube 1 and gradually diminishing amounts in tubes 2 and 3.

cytoplasmic edges of the monocyte are generally what shape

irregular and show pseudopod formation. -- These vacuoles and pseudopods are important to the overall phagocytic function of the monocytes.

Explain the significance of a CSF culture.

is a laboratory test to look for bacteria, fungi, and viruses in the normally clear fluid that moves in the space around the spinal cord.

Within the bone marrow, a nucleated red blood cell loses

its nucleus and the mature RBC is released into circulation

cytoplasm contains "ghost cells" that are

large, empty vacuoles that contain injested RBCs.

Plasma cells are ____that most lymphocytes.

larger

Segmented neutrophils, often called PMNs or segs, constitute what % WBC

largest percentage of white blood cell type. 60-70%

Malignant cells in CSF include

leukemia cells, lymphoma cells and cancer cells.

substances contained in these granules include

lysozyme, myeloperoxidase, collagenase, acid and alkaline phosphatase, in addition to many others.

Malignant cells are not normal

mature cells such as lymphocytes, neutrophils and monocytes and have morphological characteristics that are very different.

Macrophages can be found in CSF of patients with

meningitis, hemorrhage, brain abscess, infarct and contusion, and "bloody or traumatic taps".

Name 3 diseases in which the total protein is elevated.

meningitis, hemorrhage, multiple sclerosis, bacterial meningitis, tubercular meningitis

Patients with tubercular meningitis, and viral, fungal and chronic bacterial meningitis can have increased

monocytes in their cerebrospinal fluid.

cytoplasm of a monocyte color is

pale blue to grey in color and often said to have a "ground glass" appearance. It contains many small, pink-staining granules and vacuoles.

Macrophages are

phagocytic and can injest RBCs as well as other larger particles.

Neutrophils are

phagocytic and play a very important role during the inflammatory process.

Monocytes help to protect the body by

phagocytosing foreign invaders rather than mounting the cellular and humoral type responses elicited by the lymphocytes.

pink, orange, or yellow

pink, very slight hemolysis orange, heavy hemolysis or yellow, conversion of oxyhemoglobin to unconjugated bilirubin

Plasma cells function in the immune response by

producing antibodies (immunoglobulins).

The sample of malignant cells has

prominent nucleoli as well as clefting or indentation of the nucleus can also be seen in some of the cells. Variations in the cytoplasm include color, amount, and irregular edge

The macrophage nucleus stains what color

purple, is generally round, but is relatively small in size compared to the cells's abundant cytoplasm.

Metarubricyte (orthochromatic erythroblast) is the last cell stage in

red blood cell differentiation which contains a nucleus.

small lymphocyte is a little larger

than a normal red blood cell and a large lymphocyte can be up to twice the size of a red cell or even larger.

Malignant cells have generally migrated to the CSF from

the blood or other parts of the body

The color of the cytoplasm and the prominent Golgi are signs that

the cell is actively producing and packaging protein antibodies for secretion from the cell.

segmented nucleus lobes are connected by

thin filaments of chromatin. Sometimes the filaments are not apparent in the cell but they are present.

Choroid plexus cells have a very _______ appearance

uniform --and usually occur in the CSF specimen in sheets of cells.

Plasma cells are surrounded by what color cytoplasm

very dark blue cytoplasm.

metarubricyte about ready to lose its nucleus stains ______

very darkly. The nuclear chromatin material is dense.

Some of the disorders in which increased numbers of lymphocytes found in the cerebrospinal fluid are

viral, tuberculous, fungal, and syphilitic meningitis, partially treated bacterial meningitis, multiple sclerosis, and Guillain-Barre syndrome.

neutrophil contains a single nucleus

which stains dark purple or blue and which is segmented into three to five lobes.