Diagnostic Methods and Key Points of Cerebrospinal Fluid Cytology

- Reprinted article: Huashan Cerebrospinal Fluid Testing Window Original: Wang Di Huashan Cerebrospinal Fluid Testing Window

WeChat ID: hscsflab

Feature Introduction: Huashan Cerebrospinal Fluid Testing Exchange

The main issues currently facing cerebrospinal fluid cytology examinations in China are as follows: 1. Some clinicians lack sufficient awareness of the importance of cerebrospinal fluid cytology; 2. There is a lack of standardized preparation procedures (since the cell count in cerebrospinal fluid is relatively low, conventional preparation methods often yield very low detection rates; therefore, standardized preparation procedures are crucial for improving detection rates); 3. There is a severe shortage of experienced laboratory technicians and pathologists engaged in cytology examinations. Nevertheless, cerebrospinal fluid cytology remains necessary under the following circumstances: 1) Increased cerebrospinal fluid cell count; 2) Suspected subarachnoid hemorrhage with negative CT findings; 3) Screening for neoplastic meningitis; 4) MRI showing meningeal enhancement. As a rapid, effective, and low-cost method, cerebrospinal fluid cytology has become a signature diagnostic test at our hospital, serving as a screening and diagnostic tool for neurological disorders. Here, we provide a practical summary—from the processing of cerebrospinal fluid (CSF) samples through CSF cell counting and cytological classification to cytological diagnosis—for the reference and exchange of our colleagues. To gain a better understanding of the current status of CSF examinations, we have also included a brief survey on the current state of CSF testing at the end of this document. We kindly ask all colleagues to take a few minutes to complete the survey, and we welcome your valuable suggestions!

Overview

Cerebrospinal fluid (CSF) cytology is one of the most important tools available to neurologists. It involves a total cell count and cellular classification, providing crucial first-hand information about the central nervous system and the range of pathological conditions it encompasses. CSF The samples must be processed immediately, preferably within 1 hour after collection. After centrifugation, our laboratory typically performs morphological examination using Wright’s staining. When the cellular background suggests the presence of infection, we additionally perform acid-fast staining, Gram staining, and other relevant tests to identify the causative pathogens. Upon detection of atypical cells, we often employ immunocytochemical staining to further confirm and characterize the tumor cells. In normal CSF, T lymphocytes predominate, with a small number of mononuclear macrophages and occasional B lymphocytes. A marked increase in cell count, accompanied predominantly by neutrophils under microscopy, is commonly observed in bacterial meningitis and warrants further investigation for intracellular bacterial evidence. A cellular background dominated by lymphocytes and monocytes is frequently seen in viral infections and chronic inflammation. A mixed-cellular reaction pattern may occur in tuberculous meningitis. Macrophages that phagocytose red blood cells or contain fragments of hemoglobin degradation products—these latter being referred to as hemosiderin-laden macrophages—indicate old subarachnoid hemorrhage. When atypical cells suggestive of neoplasia are identified under microscopy, a comprehensive assessment combining clinical findings and immunocytochemical staining is essential for accurate diagnosis.

Processing time

Since cells in cerebrospinal fluid samples rapidly lyse after being exposed to the external environment for just two hours, unlike biochemical analysis of cerebrospinal fluid, CSF cytology and cell-count examinations require immediate processing of fresh specimens—ideally within one hour following lumbar puncture. According to our laboratory’s experience, after collection, cerebrospinal fluid can be stored at 4°C for up to 4 hours while maintaining largely intact cell morphology.

Visual inspection

Macroscopic examination of fresh CSF can provide crucial first-hand information and should be included as part of the CSF examination report. Normal cerebrospinal fluid is clear and colorless. If the white blood cell count exceeds 1,000/μL, the CSF may appear cloudy and opaque. Purulent inflammation typically presents as thick, yellow-to-green fluid. A pronounced red color suggests that the CSF sample may have been contaminated with blood. Collecting samples in consecutive tubes can help differentiate between traumatic blood contamination associated with the lumbar puncture procedure itself and true subarachnoid hemorrhage: If successive CSF samples gradually become clearer, this may indicate that the needle inadvertently injured a blood vessel during the procedure; by contrast, persistent discoloration would suggest genuine subarachnoid hemorrhage. Xanthochromia—the yellow discoloration that persists even after centrifugation—is caused by elevated bilirubin levels. It is worth noting that traumatic blood contamination can lead to an increase in the CSF cell count, and when peripheral blood contaminates the CSF, CSF cytology may show significant deviations. We have previously encountered CSF specimens in which lumbar punctures were contaminated with bone marrow cells. Therefore, it is best to obtain CSF cytology specimens in a way that minimizes such contamination. The second tube after the puncture A sample free of blood contamination.

Cell counting

Cell counts are performed using a Neubauer counting chamber. The normal range for white blood cell counts is 0 to 4 cells per microliter (mm³). A cell count exceeding 5/μL is referred to as leukocytosis or an abnormal increase in white blood cells. Immature blood-brain barrier cells exhibit higher permeability and are commonly found in cerebrospinal fluid of full-term newborns, with cell counts as high as 10/μL. In premature infants, these levels can even reach up to 30/μL. However, by the sixth week after birth, cell counts gradually decline, and any persistently elevated cell count is considered pathologically high. Based on the degree of elevation, leukocytosis can be classified as mild (5–50/μL), moderate (50–150/μL), or severe (150–3,000/μL). The following table summarizes typical cell counts and their associated pathological manifestations.

Cell counts are typically most markedly elevated in bacterial meningitis, while they show moderate elevation in chronic or viral infections. It is worth noting that... We found that in approximately one-third of patients with tumor cell dissemination into the cerebrospinal fluid, the cell count did not show a significant increase. Therefore, CSF cytology examination should be performed whenever possible, regardless of the cell count. In addition, macroscopic blood contamination inevitably leads to an increase in the CSF white blood cell count, with neutrophils being the predominant cell type observed under the microscope. In such cases, to estimate the true CSF cell count, one white blood cell should be subtracted for every 1,000–1,500 red blood cells. The CSF cell counts obtained from ventricular drainage typically show no significant difference from those obtained via lumbar puncture. In practice, while counting cell numbers using a Neubauer hemocytometer, we also perform a preliminary assessment of the size of nucleated cells in the wet smear. If we suspect the presence of abnormal cells, we prepare multiple additional cell smears for subsequent immunohistochemical staining.

Centrifugal method and conventional staining

For cytology diagnostic physicians, high-quality cytological slide preparation is crucial. After Sayk (1954) and Watson (1966) successively developed the “slide cell sedimentation method” and the “cell slide centrifugal sedimentation method,” respectively, the ability to collect more than 90% of cerebrospinal fluid cells enabled rapid advancements in this field, eventually leading to the emergence of a new discipline—cerebrospinal fluid cytology. In China, research on cerebrospinal fluid cytology began in the 1960s. Professors Hou Xide and Su Xiuchu successively established cerebrospinal fluid cytology laboratories and developed both natural sedimentation devices and miniature centrifugal sedimentation instruments for cerebrospinal fluid cell slides. In 1986, Professor Kong Fanyuan introduced the slide centrifugal sedimentation instrument and facilitated its localization. Subsequently, the Xijing Hospital of the Fourth Military Medical University further improved the original sedimentation instrument. A new-generation cell smear centrifugal sedimentation device has now been developed and put into use, achieving cell recovery and integrity rates of 80.5% and 99.9%, respectively. Building upon these advances, we have refined the cerebrospinal fluid cell collector, further enhancing both the efficiency of cell collection and the integrity of cell morphology.

Take a portion of the CSF and centrifuge it at 600–700 rpm. Specialized absorbent paper can remove the liquid components, while the cells will sediment onto a microscope slide. The conventional staining protocols include:

May-Grunwald-Giemsa (MGG)

MGG It is a standard stain used to evaluate cytological features. It consists of eosin, methylene blue, methanol, and Giemsa dyes. Typically, the cell nucleus appears darker than the cytoplasm, which tends to exhibit red, blue, or purple hues. Similarly, Wright’s stain—whose primary dyes are also eosin and methylene blue—is commonly employed in blood smears and bone marrow cytology examinations. Moreover, compared to MGG staining, Wright’s stain requires less time but yields slightly inferior staining results.

Gram staining (Gram)

Gram staining can better distinguish between Gram-positive and Gram-negative bacteria. The former appear blue to purple, while the latter appear red. Compared with other staining methods, Gram staining is typically examined using an oil immersion objective on an unmounted slide at high magnification (400× or 1000×). It should be noted that the sensitivity of Gram staining is lower than that of CSF microbial culture.

Acid-fast staining (Ziehl-Neelsen)

Acid-fast staining is indicated in cases suspected of tuberculous meningitis. It visualizes mycobacteria as bright red filamentous structures, whereas they appear poorly stained under MGG or Gram staining.

Other dyes

Other staining methods include: 1) India ink staining, used to identify Cryptococcus (characterized by small, round, dense structures surrounded by a capsule). 2) Prussian blue staining, used to detect hemosiderin—a iron-containing pigment that accumulates in macrophages following hemoglobin degradation. 3) Periodic Acid–Schiff (PAS) staining, which can help identify fungi.

Immunocytochemical labeling

In cases of suspected tumorous meningitis, immunocytochemistry can be used to detect and further characterize tumor cells. Commonly used antibodies include anti-cytokeratin (CK) and epithelial membrane antigen (EMA), both of which can confirm the presence of epithelial-derived tumor cells. Similarly, Lymphocyte markers CD3+ (T lymphocytes), CD20+ (B lymphocytes), MUM1, and PAX5 can serve as differential antibodies for the identification of B-cell tumors. When selecting immunohistochemical antibodies, we need to make a preliminary diagnosis of the disease based on the cellular morphology under Wright’s staining, and also take into account the patient’s overall clinical condition. The biggest difference between cerebrospinal fluid cell immunohistochemistry and tissue immunohistochemistry performed by pathology departments is that pathological tissues can be sliced into an unlimited number of sections, whereas the cell smears from cerebrospinal fluid are much more precious. Each antibody is selected based on the differential diagnosis of the disease.

Cerebrospinal Fluid Cytology

The following sections will provide a detailed description of the cytological morphological features observed under various conditions characterized by elevated CSF cell counts. It is best to perform CSF cytology examinations in specialized laboratories that are staffed by experienced laboratory physicians familiar with cytological characteristics.

Figure 1: Morphological features of granulocytes: (A) Normal cerebrospinal fluid: Lymphocytes and monocytes are visible. (B) Bacterial meningitis: Neutrophils predominate, with numerous intracellular and extracellular bacteria evident. (C) Eosinophilic meningitis: Characterized by distinctive binucleated eosinophils. (D) Mixed appearance involving granulocytes, lymphocytes, and monocytes. Note a single activated lymphocyte with enlarged cytoplasm and a typical perinuclear halo.

1. Normal cerebrospinal fluid contains lymphocytes and monocytes (Figure 1A). Lymphocytes typically appear round and uniform in size, with densely stained nuclear chromatin and a narrow cytoplasm that exhibits basophilic characteristics. Monocytes are larger than lymphocytes; their nuclei are oval or kidney-shaped, and their cytoplasm is broader and grayish in appearance. Occasionally, neutrophils can also be observed. Chondrocytes, which are cartilage cells, exhibit abundant eosinophilic cytoplasm and smaller nuclei with dense chromatin.

2. Bacterial Meningitis: In the absence of obvious blood contamination, the cellular morphology is predominantly characterized by neutrophils, and bacteria can be detected within the cells. The bacteria are typically deformable, with uniform shapes—either cocci or bacilli—and sometimes exhibit a halo-like appearance around them; both intracellular and extracellular bacteria may be observed. It is worth noting that occasionally, in cerebrospinal fluid samples subjected to simple secondary contamination, there may be excessive proliferation of extracellular bacteria. Gram staining can further differentiate between Gram-positive and Gram-negative bacteria. For example, Gram-positive cocci arranged in chains suggest Streptococcus pneumoniae, whereas Gram-negative cocci indicate Neisseria meningitidis. However, Gram staining often yields ambiguous results due to its tendency to produce mixed-color staining, which can lead to equivocal interpretations and reduce sensitivity. Therefore, microbial culture is considered the gold standard for bacterial identification. Moreover, CSF cultures can simultaneously undergo antimicrobial susceptibility testing, which is particularly helpful in guiding the treatment of refractory meningitis cases. In immunocompromised patients, it is important to be vigilant for acellular bacterial meningitis, which is characterized by abundant bacteria, an absent or blunted granulocytic response, and normal total cell counts.

3. Other conditions associated with elevated neutrophil percentages: Regardless of whether the absolute cell count is elevated, an increased neutrophil percentage can be observed in early stages of viral infections as well as in non-specific responses to stroke, hemorrhage, seizures, or neurosurgical procedures.

4. Diseases characterized by eosinophils: Eosinophils exhibit a typical, characteristic bilobed nucleus and abundant, densely packed eosinophilic granules in their cytoplasm (Figure 1 C). They are absent from normal cerebrospinal fluid. The predominant presence of eosinophils in clinical specimens suggests a parasitic infection or an immune response to foreign bodies, such as in cases of permanent ventricular shunting. Eosinophilic infiltration may also occasionally be observed in tuberculous or granulomatous meningitis as well as in Hodgkin’s lymphoma.

5. The mixed cellular composition is characterized by the simultaneous presence of granulocytes, lymphocytes, and monocytes, although their relative proportions may vary. This cell type is considered nonspecific and can be observed in a wide range of clinical conditions, including inflammatory and reactive states (Figure 1D). Special stains, such as acid-fast staining or PAS staining, can help differentiate between tuberculous and fungal infections; however, negative results do not rule out these diseases. Microbiological cultures can enhance diagnostic accuracy, while polymerase chain reaction (PCR) analysis is increasingly being used in clinical practice because it strikes a balance between time-consuming and costly microbiological cultures and the inexpensive but less accurate Gram staining.

6. Lymphocyte activation is characterized by significant cell enlargement, a grayish-bluish basophilic cytoplasm, and a distinctive perinuclear halo (Figure 2A). Activated lymphocytes can differentiate into mature plasma cells, which exhibit a displaced nucleus and a unique, coarsely stained chromatin structure (Figure 2B). Occasionally, binucleated forms may be observed (Figure 2C). Distinguishing between activated lymphocytes and mature plasma cells can sometimes be challenging, as intermediate differentiation stages are common and reflect ongoing cellular differentiation. Both cell types can be found in viral or autoimmune-related diseases, as well as in response to nonspecific meningeal irritation—including chemical or mechanical noxious stimuli. In neurolymphomatosis or herpesvirus infections, excessively activated lymphocytes may be difficult to differentiate from malignant lymphoma cells (Figure 2D). Malignant lymphoproliferative disorders are typically characterized by a homogeneous, monoclonal population of cells; however, the presence of more heterogeneous lymphocytes of varying sizes, along with mature plasma cells, supports a diagnosis of inflammatory infection. Additionally, detection of polyclonal intramedullary immunoglobulins can serve as an aid in further differential diagnosis of malignancies. Immunocytochemistry and/or fluorescence-activated cell sorting, combined with detailed immunophenotypic analysis of cell populations, represent further useful tools for assessing cellular clonality.

Figure 2: Various morphological features of lymphocytes: (A) Lymphocytes ranging in size from small to large. (B) Plasma cells with characteristic coarse chromatin structure and cytoplasmic displacement. (C) Occasionally observed binucleated plasma cells (indicated by arrows). (D) Highly activated lymphocytes, including large lymphocytes resembling lymphoma cells and lymphocytes showing mitotic figures (indicated by arrows).

7. Differential Diagnosis Between Subarachnoid Hemorrhage and Fresh Blood Contamination: Unlike fresh blood contamination (Figure 3A), the presence of erythrophages and hemosiderophages tends to suggest a previous diagnosis of subarachnoid hemorrhage (Figures 3B and 3C). Both cell types are macrophages—derived from vacuolated, activated monocytes—that have phagocytosed red blood cells and blue-colored, iron-containing hemosiderin formed after hemoglobin degradation. It is worth noting that isolated hemosiderophages may also be encountered during repeated lumbar punctures, arising from the trauma caused by the lumbar puncture procedure itself. Bilirubin crystals, resulting from bilirubin accumulation, appear as small, yellow-to-orange crystals found either outside cells or within hemosiderophages (Figure 3D).

Figure 3: Characteristics of Hemorrhagic Disorders: (A) Fresh blood contamination, with dense red blood cells and occasional neutrophils. (B) Erythrophagocytosis—clusters of cells with extensively vacuolated cytoplasm, in which phagocytosed red blood cell fragments are visible. (C) Iron-phagocytosing cells—blue-colored cells with foamy cytoplasm containing hemosiderin granules. (D) Bilirubin crystals—surrounded by red blood cells, yellow in color, with bilirubin crystals indicated by arrows.

8. Manifestations of neoplastic meningitis: Tumor cells typically exhibit large, irregularly shaped nuclei rich in chromatin, with prominent nucleoli and dark, basophilic cytoplasm (Figure 4A). The nuclear-to-cytoplasmic ratio is usually high. In adenocarcinoma cells, the cytoplasm often contains large vacuoles—vesicles that store mucin. Sometimes, due to the large size of these vesicles, the nucleus becomes displaced, a feature known as signet-ring cells. Some cancer cells display irregular cytoplasmic protrusions and pseudopodia (Figure 4C). Similarly, numerous mitotic figures also suggest a neoplastic origin, whereas isolated mitoses are occasionally observed in normal or inflammatory-reactive cells (Figure 4C). A dense population of relatively uniform-shaped cells suggests lymphoma (Figure 4D). Immunohistochemical markers—such as keratin staining for epithelial-derived carcinomas and hematological markers—can further facilitate precise subtype analysis (Figures 4B, D, and F).

The main issues currently facing cerebrospinal fluid cytology examinations in China are as follows: 1. Some clinicians lack sufficient awareness of the importance of cerebrospinal fluid cytology; 2. There is a lack of standardized preparation procedures (since the cell count in cerebrospinal fluid is relatively low, conventional preparation methods often yield very low detection rates; therefore, standardized preparation procedures are crucial for improving detection rates); 3. There is a severe shortage of experienced laboratory technicians and pathologists engaged in cytology examinations. Nevertheless, cerebrospinal fluid cytology remains necessary under the following circumstances: 1) Increased cerebrospinal fluid cell count; 2) Suspected subarachnoid hemorrhage with negative CT findings; 3) Screening for neoplastic meningitis; 4) MRI showing meningeal enhancement. As a rapid, effective, and low-cost method, cerebrospinal fluid cytology has become a signature diagnostic test at our hospital, serving as a screening and diagnostic tool for neurological disorders. Here, we provide a practical summary—from the processing of cerebrospinal fluid (CSF) samples through CSF cell counting and cytological classification to cytological diagnosis—for the reference and exchange of our colleagues. To gain a better understanding of the current status of CSF examinations, we have also included a brief survey on the current state of CSF testing at the end of this document. We kindly ask all colleagues to take a few minutes to complete the survey, and we welcome your valuable suggestions!

Overview

Cerebrospinal fluid (CSF) cytology is one of the most important tools available to neurologists. It involves a total cell count and cellular classification, providing crucial first-hand information about the central nervous system and the range of pathological conditions it encompasses. CSF The samples must be processed immediately, preferably within 1 hour after collection. After centrifugation, our laboratory typically performs morphological examination using Wright’s staining. When the cellular background suggests the presence of infection, we additionally perform acid-fast staining, Gram staining, and other relevant tests to identify the causative pathogens. Upon detection of atypical cells, we often employ immunocytochemical staining to further confirm and characterize the tumor cells. In normal CSF, T lymphocytes predominate, with a small number of mononuclear macrophages and occasional B lymphocytes. A marked increase in cell count, accompanied predominantly by neutrophils under microscopy, is commonly observed in bacterial meningitis and warrants further investigation for intracellular bacterial evidence. A cellular background dominated by lymphocytes and monocytes is frequently seen in viral infections and chronic inflammation. A mixed-cellular reaction pattern may occur in tuberculous meningitis. Macrophages that phagocytose red blood cells or contain fragments of hemoglobin degradation products—these latter being referred to as hemosiderin-laden macrophages—indicate old subarachnoid hemorrhage. When atypical cells suggestive of neoplasia are identified under microscopy, a comprehensive assessment combining clinical findings and immunocytochemical staining is essential for accurate diagnosis.

Processing time

Since cells in cerebrospinal fluid samples rapidly lyse after being exposed to the external environment for just two hours, unlike biochemical analysis of cerebrospinal fluid, CSF cytology and cell-count examinations require immediate processing of fresh specimens—ideally within one hour following lumbar puncture. According to our laboratory’s experience, after collection, cerebrospinal fluid can be stored at 4°C for up to 4 hours while maintaining largely intact cell morphology.

Visual inspection

Macroscopic examination of fresh CSF can provide crucial first-hand information and should be included as part of the CSF examination report. Normal cerebrospinal fluid is clear and colorless. If the white blood cell count exceeds 1,000/μL, the CSF may appear cloudy and opaque. Purulent inflammation typically presents as thick, yellow-to-green fluid. A pronounced red color suggests that the CSF sample may have been contaminated with blood. Collecting samples in consecutive tubes can help differentiate between traumatic blood contamination associated with the lumbar puncture procedure itself and true subarachnoid hemorrhage: If successive CSF samples gradually become clearer, this may indicate that the needle inadvertently injured a blood vessel during the procedure; by contrast, persistent discoloration would suggest genuine subarachnoid hemorrhage. Xanthochromia—the yellow discoloration that persists even after centrifugation—is caused by elevated bilirubin levels. It is worth noting that traumatic blood contamination can lead to an increase in the CSF cell count, and when peripheral blood contaminates the CSF, CSF cytology may show significant deviations. We have previously encountered CSF specimens in which lumbar punctures were contaminated with bone marrow cells. Therefore, it is best to obtain CSF cytology specimens in a way that minimizes such contamination. The second tube after the puncture A sample free of blood contamination.

Cell counting

Cell counts are performed using a Neubauer counting chamber. The normal range for white blood cell counts is 0 to 4 cells per microliter (mm³). A cell count exceeding 5/μL is referred to as leukocytosis or an abnormal increase in white blood cells. Immature blood-brain barrier cells exhibit higher permeability and are commonly found in cerebrospinal fluid of full-term newborns, with cell counts as high as 10/μL. In premature infants, these levels can even reach up to 30/μL. However, by the sixth week after birth, cell counts gradually decline, and any persistently elevated cell count is considered pathologically high. Based on the degree of elevation, leukocytosis can be classified as mild (5–50/μL), moderate (50–150/μL), or severe (150–3,000/μL). The following table summarizes typical cell counts and their associated pathological manifestations.

Cell counts are typically most markedly elevated in bacterial meningitis, while they show moderate elevation in chronic or viral infections. It is worth noting that... We found that in approximately one-third of patients with tumor cell dissemination into the cerebrospinal fluid, the cell count did not show a significant increase. Therefore, CSF cytology examination should be performed whenever possible, regardless of the cell count. In addition, macroscopic blood contamination inevitably leads to an increase in the CSF white blood cell count, with neutrophils being the predominant cell type observed under the microscope. In such cases, to estimate the true CSF cell count, one white blood cell should be subtracted for every 1,000–1,500 red blood cells. The CSF cell counts obtained from ventricular drainage typically show no significant difference from those obtained via lumbar puncture. In practice, while counting cell numbers using a Neubauer hemocytometer, we also perform a preliminary assessment of the size of nucleated cells in the wet smear. If we suspect the presence of abnormal cells, we prepare multiple additional cell smears for subsequent immunohistochemical staining.

Centrifugal method and conventional staining

For cytology diagnostic physicians, high-quality cytological slide preparation is crucial. After Sayk (1954) and Watson (1966) successively developed the "slide cell sedimentation method" and the "cell slide centrifugal sedimentation method," respectively, the ability to collect more than 90% of cerebrospinal fluid cells enabled rapid advancements in this field, eventually leading to the emergence of a new discipline—cerebrospinal fluid cytology. In China, research on cerebrospinal fluid cytology began in the 1960s. Professors Hou Xide and Su Xiuchu successively established cerebrospinal fluid cytology laboratories and developed the Hou-type natural sedimentation device as well as the Su-type FMU-5 miniature cerebrospinal fluid cell slide centrifugal sedimentation apparatus. In 1986, Professor Kong Fanyuan introduced the Cytospin-2 slide centrifugal sedimentation apparatus and facilitated its localization. Subsequently, the Xijing Hospital of the Fourth Military Medical University further improved the original Su-type sedimentation apparatus. Today, the next-generation FMU-6 cell smear centrifugal sedimentation apparatus has been developed and put into use, achieving cell recovery and integrity rates of 80.5% and 99.9%, respectively. Building upon these advances, we have refined the cerebrospinal fluid cell collector, further enhancing both the efficiency of cerebrospinal fluid cell collection and the integrity of cell morphology.

Take a portion of the CSF and centrifuge it at 600–700 rpm. Specialized absorbent paper can remove the liquid components, while the cells will sediment onto a microscope slide. The conventional staining protocols include:

May-Grunwald-Giemsa (MGG)

MGG It is a standard stain used to evaluate cytological features. It consists of eosin, methylene blue, methanol, and Giemsa dyes. Typically, the cell nucleus appears darker than the cytoplasm, which tends to exhibit red, blue, or purple hues. Similarly, Wright’s stain—whose primary dyes are also eosin and methylene blue—is commonly employed in blood smears and bone marrow cytology examinations. Moreover, compared to MGG staining, Wright’s stain requires less time but yields slightly inferior staining results.

Gram staining (Gram)

Gram staining can better distinguish between Gram-positive and Gram-negative bacteria. The former appear blue to purple, while the latter appear red. Compared with other staining methods, Gram staining is typically examined using an oil immersion objective on an unmounted slide at high magnification (400× or 1000×). It should be noted that the sensitivity of Gram staining is lower than that of CSF microbial culture.

Acid-fast staining (Ziehl-Neelsen)

Acid-fast staining is indicated in cases suspected of tuberculous meningitis. It visualizes mycobacteria as bright red filamentous structures, whereas they appear poorly stained under MGG or Gram staining.

Other dyes

Other staining methods include: 1) India ink staining, used to identify Cryptococcus (characterized by small, round, dense structures surrounded by a capsule). 2) Prussian blue staining, used to detect hemosiderin—a iron-containing pigment that accumulates in macrophages following hemoglobin degradation. 3) Periodic Acid–Schiff (PAS) staining, which can help identify fungi.

Immunocytochemical labeling

In cases of suspected tumorous meningitis, immunocytochemistry can be used to detect and further characterize tumor cells. Commonly used antibodies include anti-cytokeratin (CK) and epithelial membrane antigen (EMA), both of which can confirm the presence of epithelial-derived tumor cells. Similarly, Lymphocyte markers CD3+ (T lymphocytes), CD20+ (B lymphocytes), MUM1, and PAX5 can serve as differential antibodies for the identification of B-cell tumors. When selecting immunohistochemical antibodies, we need to make a preliminary diagnosis of the disease based on the cellular morphology under Wright’s staining, and also take into account the patient’s overall clinical condition. The biggest difference between cerebrospinal fluid cell immunohistochemistry and tissue immunohistochemistry performed by pathology departments is that pathological tissues can be sliced into an unlimited number of sections, whereas the cell smears from cerebrospinal fluid are much more precious. Each antibody is selected based on the differential diagnosis of the disease.

Cerebrospinal Fluid Cytology

The following sections will provide a detailed description of the cytological morphological features observed under various conditions characterized by elevated CSF cell counts. It is best to perform CSF cytology examinations in specialized laboratories that are staffed by experienced laboratory physicians familiar with cytological characteristics.

Figure 1: Morphological features of granulocytes: (A) Normal cerebrospinal fluid: Lymphocytes and monocytes are visible. (B) Bacterial meningitis: Neutrophils predominate, with numerous intracellular and extracellular bacteria evident. (C) Eosinophilic meningitis: Characterized by distinctive binucleated eosinophils. (D) Mixed appearance involving granulocytes, lymphocytes, and monocytes. Note a single activated lymphocyte with enlarged cytoplasm and a typical perinuclear halo.

1. Normal cerebrospinal fluid contains lymphocytes and monocytes (Figure 1A). Lymphocytes typically appear round and uniform in size, with densely stained nuclear chromatin and a narrow cytoplasm that exhibits basophilic characteristics. Monocytes are larger than lymphocytes; their nuclei are oval or kidney-shaped, and their cytoplasm is broader and grayish in appearance. Occasionally, neutrophils can also be observed. Chondrocytes, which are cartilage cells, exhibit abundant eosinophilic cytoplasm and smaller nuclei with dense chromatin.

2. Bacterial Meningitis: In the absence of obvious blood contamination, the cellular morphology is predominantly characterized by neutrophils, and bacteria can be detected within the cells. The bacteria are typically deformable, with uniform shapes—either cocci or bacilli—and sometimes exhibit a halo-like appearance around them; both intracellular and extracellular bacteria may be observed. It is worth noting that occasionally, in cerebrospinal fluid samples subjected to simple secondary contamination, there may be excessive proliferation of extracellular bacteria. Gram staining can further differentiate between Gram-positive and Gram-negative bacteria. For example, Gram-positive cocci arranged in chains suggest Streptococcus pneumoniae, whereas Gram-negative cocci indicate Neisseria meningitidis. However, Gram staining often yields ambiguous results due to its tendency to produce mixed-color staining, which can lead to equivocal interpretations and reduce sensitivity. Therefore, microbial culture is considered the gold standard for bacterial identification. Moreover, CSF cultures can simultaneously undergo antimicrobial susceptibility testing, which is particularly helpful in guiding the treatment of refractory meningitis cases. In immunocompromised patients, it is important to be vigilant for acellular bacterial meningitis, which is characterized by abundant bacteria, an absent or blunted granulocytic response, and normal total cell counts.

3. Other conditions associated with elevated neutrophil percentages: Regardless of whether the absolute cell count is elevated, an increased neutrophil percentage can be observed in early stages of viral infections as well as in non-specific responses to stroke, hemorrhage, seizures, or neurosurgical procedures.

4. Diseases characterized by eosinophils: Eosinophils exhibit a typical, characteristic bilobed nucleus and abundant, densely packed eosinophilic granules in their cytoplasm (Figure 1 C). They are absent from normal cerebrospinal fluid. The predominant presence of eosinophils in clinical specimens suggests a parasitic infection or an immune response to foreign bodies, such as in cases of permanent ventricular shunting. Eosinophilic infiltration may also occasionally be observed in tuberculous or granulomatous meningitis as well as in Hodgkin’s lymphoma.

5. The mixed cellular composition is characterized by the simultaneous presence of granulocytes, lymphocytes, and monocytes, although their relative proportions may vary. This cell type is considered nonspecific and can be observed in a wide range of clinical conditions, including inflammatory and reactive states (Figure 1D). Special stains, such as acid-fast staining or PAS staining, can help differentiate between tuberculous and fungal infections; however, negative results do not rule out these diseases. Microbiological cultures can enhance diagnostic accuracy, while polymerase chain reaction (PCR) analysis is increasingly being used in clinical practice because it strikes a balance between time-consuming and costly microbiological cultures and the inexpensive but less accurate Gram staining.