Cerebrospinal Fluid Analysis

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Cerebrospinal Fluid Analysis

Learning Objectives

Key Terms1

Physiology and Composition

Cerebrospinal fluid (CSF) bathes the brain and spinal cord. CSF is produced primarily (70%) from secretions into the four ventricles of the brain by the highly vascular choroid plexus (vascular fringe–like folds in the pia mater). The ependymal cells that line the brain and spinal cord also play a minor role in the production of CSF. The formation of CSF can be described as a selective secretion from plasma, not as an ultrafiltrate. This is evidenced by higher CSF concentrations of some solutes (e.g., sodium, chloride, magnesium) and lower CSF concentrations of other solutes (e.g., potassium, total calcium) compared to plasma. If simple ultrafiltration were responsible for CSF production, these solute concentration differences would not exist.

The brain and spinal cord are surrounded by three membranes, collectively termed the meninges. The tough outermost membrane, the dura mater, is next to the bone. The arachnoid (also called arachnoidea), or middle layer, derives its name from its visual resemblance to a spider web. The innermost membrane, the pia mater, adheres to the surface of the neural tissues (Fig. 9.1). Cerebrospinal fluid flows in the space between the arachnoidea mater and the pia mater, called the subarachnoid space, where it bathes and protects the delicate tissues of the central nervous system (CNS). From its initial formation in the ventricles, the CSF circulates to the brainstem and spinal cord, principally through pressure changes caused by postural, respiratory, and circulatory pressures (Fig. 9.2). The CSF eventually flows in the subarachnoid space to the top outer surface of the brain, where projections of the arachnoid membrane called arachnoid granulations are present. These projections have small, one-way valvelike structures that allow the CSF to enter the bloodstream of the large veins of the head. Cerebrospinal fluid formation, circulation, and reabsorption into the blood make up a dynamic process that constantly turns over about 20 mL each hour.1 If the flow path between production and reabsorption of CSF into the blood is obstructed for any reason, CSF accumulates, producing hydrocephalus; intracranial pressure can increase, causing brain damage, intellectual and developmental disabilities, or death if left untreated. Normally, the total volume of CSF in an adult ranges from 85 to 150 mL. The volume in neonates is significantly smaller, ranging from 10 to 60 mL.

The CSF protects and supports the brain and spinal cord and provides a medium for the transport and exchange of nutrients and metabolic wastes. The capillary endothelium in contact with CSF enables the transfer of substances from the blood into the CSF and vice versa. This capillary endothelium differs from the endothelium in other tissues by the presence of tight junctions between adjacent endothelial cells. These tight junctions significantly reduce the extracellular passage of substances from the blood plasma into the CSF. In other words, all substances that enter or leave the CSF must pass through the membranes and cytoplasm of the capillary endothelial cells. This modulating interface between the blood and the CSF is called the blood-brain barrier and accounts for the observed concentration differences of electrolytes, proteins, and other solutes. An example of the selectivity and effectiveness of this blood-brain barrier is the failure of some antibiotics (e.g., penicillins), given intravenously, to enter the CSF, although these antibiotics freely penetrate all other tissues of the body.

In healthy individuals, the chemical composition of CSF is closely regulated and includes low-molecular-weight proteins. Changes in the chemical composition or in the cellular components can aid in the diagnosis of disease. Protein, glucose, and lactate are routinely measured in CSF. Although numerous other parameters (e.g., sodium, potassium, chloride, magnesium, pH, PCO2, enzymes) have been evaluated for clinical use, they have yet to prove their diagnostic value. In addition to chemical analysis, CSF is routinely cultured for microbial organisms, examined microscopically to evaluate the cellular components, and tested for the presence of specific antigens. These cytologic, microbiological, and immunologic studies can provide valuable diagnostic information. For CSF reference intervals, see Table 9.1 or Appendix C.

Table 9.1

Cerebrospinal Fluid Reference Intervalsa
Physical Examination
Color Colorless
Clarity Clear

Chemical Examination
Component Conventional Units Conversion Factor SI Units
 Calcium 2.0–2.8 mEq/L 0.5 1.00–1.40 mmol/L
 Chloride 115–130 mEq/L 1 115–130 mmol/L
 Lactate 10–22 mg/dL 0.111 1.1–2.4 mmol/L
 Magnesium 2.4–3.0 mEq/L 0.5 1.2–1.5 mmol/L
 Potassium 2.6–3.0 mEq/L 1 2.6–3.0 mmol/L
 Sodium 135–150 mEq/L 1 135–150 mmol/L
Glucose 50–80 mg/dL 0.5551 2.8–4.4 mmol/L
Total protein 15–45 mg/dL 10 150–450 mg/L
Albumin 10–30 mg/dL 10 100–300 mg/L
IgG 1–4 mg/dL 10 10–40 mg/L

Protein Electrophoresis Percent of Total Protein
Transthyretin (prealbumin) 2–7%
Albumin 56–76%
α1-Globulin 2–7%
α2-Globulin 4–12%
β-Globulin 8–18%
γ-Globulin 3–12%

Microscopic Examination
Component Conventional Units Conversion Factor SI Units
Neonates (<1 year old) 0–30 cells/μL 106 0–30 × 106 cells/L
1–4 years old 0–20 cells/μL 106 0–20 × 106 cells/L
5–18 years old 0–10 cells/μL 106 0–10 × 106 cells/L
Adults (>18 years old) 0–5 cells/μL 106 0–5 × 106 cells/L

Differential Cell Count Percent of Total Count
 Lymphocytes 5–35%
 Monocytes 50–90%
 Neutrophils 0–8%
 Lymphocytes 40–80%
 Monocytes 15–45%
 Neutrophils 0–6%


aFor cerebrospinal fluid specimens obtained by lumbar puncture.

Specimen Collection

Cerebrospinal fluid specimens are collected specifically for the diagnosis or treatment of disease (Box 9.1). Although the lumbar puncture principally used to obtain CSF specimens is fairly routine, it involves significant patient discomfort and can cause complications. Therefore once a CSF specimen has been collected, it is imperative that it is properly labeled and handled at the bedside and in the laboratory.

Usually a physician performs a lumbar puncture in the third or fourth lumbar interspace (or lower) in adults or in the fourth or fifth interspace in children (Fig. 9.3). The puncture site selection can vary if an infection is present at the preferred site. A locally infected site must be avoided to prevent introduction of the infection into the CNS. The lumbar puncture procedure is performed aseptically after thorough cleansing of the patient’s skin and the application of a local anesthetic. The spinal needle is advanced into the lumbar interspace, and often a “pop” is heard on penetration of the dura mater. Immediately after the dura mater has been entered and before any CSF has been removed, the physician takes the initial or “opening” pressure of the CSF using a manometer that attaches to the spinal needle. Normal CSF pressures for an adult in a lateral recumbent position range from 50 to 180 mm Hg, with slightly higher pressures obtained from individuals in a sitting position. If the pressure is in the normal range, up to 20 mL of CSF (approximately 15% of the estimated total CSF volume) can be removed safely. If the CSF pressure is less than or greater than normal, only 1 to 2 mL should be removed. Because the total volume of CSF is significantly smaller in infants and children, proportionately smaller volumes are collected from them. After the CSF has been removed and before the spinal needle has been withdrawn, the physician takes the “closing” CSF pressure, which should be 10 to 30 mm Hg less than the opening pressure. Both CSF pressure values and the amount of CSF removed are recorded in the patient’s chart.

As CSF is collected, it is dispensed into three (or more) sequentially labeled sterile collection tubes. The first tube is used for chemical and immunologic testing, because any minimal blood contamination resulting from vessel injury during the initial tap normally does not affect these results. The second tube is used for microbial testing, and the third tube is reserved for the microscopic examination of cellular components (i.e., red and white blood cell counts and cytologic studies). If only a small amount of CSF is obtained and a single collection tube must be used, the ordering physician prioritizes the tests desired. With these low-volume specimens, it is imperative that a portion of the specimen is maintained sterile when microbiology tests are ordered. This can be achieved by having the microbiology laboratory receive and process the specimen first, or, by using sterile technique, a sufficient volume of the specimen is transferred to another container for cell counts and any chemical or immunologic tests requested.

The examination and testing of CSF should take place as soon as possible after collection. Therefore in most institutions, tests ordered on CSF specimens are considered stat. Delay in testing can cause inaccurate results, such as falsely low cell counts caused by the lysis of white blood cells or falsely high lactate levels caused by glycolysis. In addition, the recovery of viable microbial organisms is jeopardized. When delay is unavoidable, each CSF collection tube must be stored at the temperature that best ensures recovery of the constituents of interest (Table 9.2). Any CSF remaining after the initial tests have been performed can be frozen and saved for possible future chemical or immunologic studies.

Table 9.2

Cerebrospinal Fluid Specimen Handling and Storage Temperature
Tube #1 Chemical, immunology, serology Frozen (−15 to −30°C)
Tube #2 Microbiological studies Room temperature (19–26°C)
Tube #3 Cell counts and cytology studies Refrigerated (2–8°C)

Physical Examination

Normal CSF is clear and colorless, with a viscosity similar to that of water. Increased viscosity, although rare, can occur as a result of metastatic, mucin-secreting adenocarcinomas. Abnormally increased amounts of fibrinogen in CSF caused by a compromised blood-brain barrier can result in clot formation. Fine, delicate clots can form a thin film or pellicle on the surface of CSF after it has been stored at refrigerator temperatures for 12 or more hours. Most often, clot formation is associated with a traumatic puncture procedure, in which blood and plasma proteins contaminated the CSF. Rarely, no blood is present in the CSF, and clots form as a result of elevated CSF protein levels with conditions such as Froin’s syndrome or suppurative or tuberculous meningitis, or as a result of subarachnoid obstruction. Despite the various possibilities for clot formation, clots rarely are encountered, even in patients with pathologic conditions. If present, however, clot formation must be noted and reported.

Because a CSF specimen is collected into three or more collection tubes and all tubes may not be sent to the same laboratory area, the testing personnel must examine and individually assess each tube for clarity, color, and volume. The clarity or turbidity of CSF depends on its cellularity. Pleocytosis, an increase in the number of cells in CSF, causes the CSF to appear cloudy to varying degrees. A cloudy CSF specimen is associated with a white blood cell count greater than 200 cells/μL or a red blood cell count exceeding 400 cells/μL. Similarly, microorganisms or an increased protein content can produce cloudy CSF specimens. Cerebrospinal fluid clarity can be graded semiquantitatively from 0 (clear) to 4+ (newsprint cannot be read through the fluid) using standardized criteria to ensure consistency in reporting. Occasionally, the CSF may appear oily because of the presence of radiographic contrast media.

Although normal CSF is colorless, in disease states it often appears xanthochromic. Although xanthochromia literally means a yellow discoloration, this term is applied to a spectrum of CSF discolorations, including pink, orange, and yellow. A pink supernatant after centrifugation results from oxyhemoglobin, a yellow supernatant results from bilirubin, an orange supernatant results from a combination of these, and a brownish supernatant results from methemoglobin formation. High concentrations of other substances, such as carotene, and protein in concentrations greater than 150 mg/dLcan cause xanthochromic CSF specimens, as can conditions such as meningeal melanoma or collection of the CSF 2 to 5 days after a traumatic tap (Box 9.2).

Hemorrhage or Traumatic Tap

Gross blood in CSF is visually apparent, and determining its source requires differentiation between a traumatic puncture procedure and a subarachnoid or intracerebral hemorrhage. Several observations can aid in making this differentiation (Table 9.3). First, a traumatic tap results in the greatest amount of blood collected in the first specimen tube. Hence a visual assessment or a comparison of the RBC count between tube 1 and tube 3 (or 4) will show a significant difference (decrease). In contrast, a hemorrhage results in a homogeneous distribution of RBCs throughout all collection tubes.

Table 9.3

Features that Aid in Differentiating Traumatic Tap from Hemorrhage
Traumatic Tap Hemorrhage
Amount of blood decreases or clears progressively from first to last collection tube Amount of blood the same in all collection tubes
Streaking of blood in CSF during collection Blood evenly dispersed during collection
CSF may clot CSF does not clot due to defibrination in vivo
Usually no xanthochromia Xanthochromia present
No hemosiderin present Presence of hemosiderin-laden macrophages (siderophage)

CSF, Cerebrospinal fluid.

Second, after centrifugation of CSF, when the supernate is colorless, it points to a traumatic tap. In contrast, a xanthochromic supernate indicates a previous hemorrhage. It takes 1 to 2 hours for RBC lysis to occur in CSF and for the formation of the compounds that produce xanthochromia.2 The lysis of RBCs observed in CSF is not osmotically induced because plasma and CSF are osmotically equivalent; rather, it is speculated that the lack of sufficient CSF proteins and lipids needed to stabilize RBC membranes causes the lysis. Because lysis can occur in vivo or in vitro, timely processing and testing of CSF specimens are necessary. Once RBC lysis has occurred in CSF, xanthochromia can be evident as early as 2 hours after the hemorrhage and persist for as long as 4 weeks.3

Lastly, when the microscopic examination of CSF reveals macrophages with phagocytosed RBCs (erythrophages), it indicates either a hemorrhage or a previous traumatic tap. As hemoglobin from RBCs is processed within macrophages, they become siderophages (i.e., cells with intracellular hemosiderin granules), and hematin crystals may be present. See subsection Macrophages later in this chapter for additional discussion and a timeline of the cellular findings in CSF following a hemorrhage or traumatic tap.

Microscopic Examination

The CSF of adults normally contains a small number of white blood cells (WBCs), specifically, lymphocytes and monocytes at 0 to 5 cells/μL. Similarly low numbers of WBCs (0–10 cells/μL) are expected in children, whereas healthy neonates can have up to 30 WBCs/μL, with monocytes predominating.

In contrast, RBCs are not normally present in CSF. When present, RBCs most often represent CSF contamination with peripheral blood during the lumbar puncture procedure. Rarely, RBCs are present because of a recent (within 1 or 2 hours) subarachnoid or cerebral hemorrhage.

Cell counts on CSF must be performed as soon as possible to ensure valid results. At room temperature, 40% of the WBCs in CSF will lyse in 2 hours.4 If the specimen is refrigerated, WBC lysis can be reduced significantly to approximately 15%, but not completely prevented. Similarly, RBCs do not demonstrate significant lysis at 4°C; therefore the CSF collection tube for cell counts should be refrigerated if the count must be delayed for any reason.

Depending on the testing institution, different approaches to CSF cell counts are possible. Some laboratories do not perform a total cell count; instead, they perform individual RBC and WBC counts. The sum of these two counts is equivalent to a total cell count. Other laboratories perform a total cell count and a WBC count; the difference between these counts is the RBC count.

Total Cell Count

Despite being labor intensive and technically challenging with low precision, cell counts on CSF are often performed manually. Chapter 17 describes a manual procedure for performing CSF and other cell counts using a hemacytometer, and Appendix D provides details for preparing the various diluents that can be used. Commercial control materials are available to monitor the technical performance of personnel performing manual hemacytometer cell counts. However, it is possible to prepare “in-house” simulated CSF specimens using the method developed by Lofsness and Jensen.5 These simulated specimens can be used (1) as quality control samples, (2) as samples for training, or (3) for competency assessment of laboratory personnel.

Total cell counts on CSF are usually made using well-mixed, undiluted CSF. Because of the low viscosity and protein content of CSF, cells settle within 1 minute after filling the hemacytometer chambers. When counting clear CSF, the standard of practice is to count all nine large squares on both sides of the hemacytometer. When the number of cells in the nine squares exceeds 200 or is crowded or overlapping, the CSF should be diluted with isotonic saline. Dilutions vary according to the concentrations of cells present. Sometimes an initial dilution may be selected based on the visual appearance of the fluid, ranging from a 1:10 dilution for a slightly cloudy specimen to a 1:10,000 dilution for bloody specimens.

Automation of CSF cell counts significantly increases analytical precision and reduces turnaround time. However, because the number of cells in CSF is normally low (<5 cells/μLin adults or <30 cells/μL in children), many electronic impedance-based cell counters cannot be used because the instrument’s background count produces values higher than these “normal” cell counts. This is an issue, especially when most of the CSF samples actually tested in the laboratory have low cell counts that reside in the normal range.

Some automated systems currently available for CSF cell counting are the ADVIA 120 and ADVIA 2120 hematology analyzers (Siemens Healthcare Diagnostics, Deerfield, IL), Sysmex XE-5000 hematology analyzers (Sysmex Corporation, Mundelein, IL), and the Iris iQ200 with Body Fluids Module (Beckman Coulter Inc., Brea, CA). Studies using these analyzers indicate agreement with the manual hemacytometer method, and the best correlations are obtained at high cell counts.68 The ADVIA and Sysmex analyzers enumerate and differentiate cells using flow cytometry, whereas the iQ200 enumerates and differentiates RBCs and nucleated cells using flow cell digital imaging. Note that automated systems are not used to differentiate nucleated cells or to identify pathologic cell types such as neoplastic cells, siderophages, and lipophages. (See Chapter 16for additional discussion of automated body fluid analysis.)

Red Blood Cell (Erythrocyte) Count

RBC counts provide little diagnostically useful information. They can be performed to aid in the differentiation of a recent hemorrhage from a traumatic puncture procedure, as previously discussed. Another application of the RBC count is to correct the WBC count and total protein determinations obtained from a CSF specimen known to be contaminated with peripheral blood. These calculated “corrections” have limited accuracy, usually overcorrect the counts, assume that all of the RBCs present result from contamination, and have little clinical use. Therefore this chapter does not describe these corrections in detail; readers are referred to the bibliography for additional information.

As with the total cell count, well-mixed, undiluted CSF is used for the RBC count unless the number of cells present requires dilution because of overlapping and crowding of cells. Because the differentiation between small lymphocytes and crenated erythrocytes can be difficult in unstained wet preparations, some laboratories eliminate this count, replacing it with the difference obtained between the total cell count and the WBC count.

White Blood Cell (Leukocyte) Count

Increased WBC counts in CSF are associated with diseases of the CNS and a variety of other conditions (Table 9.4). The WBC count can vary significantly depending on the causative agent. Often the highest WBC counts (greater than 50,000 cells/μL) in CSF occur with bacterial meningitis. However, the same condition can show no pleocytosis in some patients.9

Table 9.4

Cell Types and Causes of Cerebrospinal Fluid Pleocytosis
Predominant Cell Type Infectious Causes Noninfectious Causes
Neutrophils Meningitis
Allergic reaction to
Lymphocytes Meningitis

Plasma cells Same disorders associated with increased lymphocytes, particularly tuberculous and syphilitic meningitis
Monocytes Meningitis
Response to RBCs or lipids inCSF resulting from:

Malignant cells

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Oct 18, 2022 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Cerebrospinal Fluid Analysis

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