Body Fluid Examination

Body Fluid Examination

Professor Avery Tibbs, MLS(ASCP)CM

Objectives

• List the types of body fluids from closed body cavities that are studied in the hematology laboratory.

• Identify the type of procedure used to obtain each type of body fluid.

• Evaluate the formation of serous (peritoneal, pericardial, and pleural), cerebrospinal, and synovial fluids.

• Assess the cell types normally found in serous, cerebrospinal, and synovial fluids.

• Analyze effusion, transudate, and exudate and their characteristics.

• Assess the laboratory methods for body fluid analysis.

• Evaluate the principle, advantages, and disadvantages of the cytocentrifuge method for body fluid morphological examination.

• Describe the common cellular artifacts introduced by cytocentrifugation.

Introduction

• Analysis of body fluid from normally sterile body compartments is an important part of diagnosis and treatment.

• Hematology laboratory is responsible for:

  • Cell counts.

  • Morphological examination.

  • Cell differentiation of body fluids.

• Types of cells found in these fluids include:

  • White Blood Cells (WBCs).

  • Red Blood Cells (RBCs).

  • Tissue cells.

  • Tumor cells.

• Differentiation between benign and malignant cells is key for accurate pathology referrals.

• Diagnostically significant crystals may also form in fluids.

Types of Body Fluids

• Definition: Body fluid is an all-inclusive term for all nonblood, nonurine fluids from closed body cavities.

  • Locations include:

    • Thoracic and abdominal cavities.

    • Central Nervous System (CNS).

    • Joint spaces.

• Types of fluids:

  • Serous fluids: All fluids from the thoracic and abdominal spaces.

  • Cerebrospinal fluid (CSF): Found within the spinal column and surrounding the brain.

  • Synovial fluid: Found within all joints of the body.

• All fluids are ultrafiltrates of plasma and are normally cell-free.

Serous Fluids

• Membranes surround the heart, lungs, and abdominal cavity, creating individual cavities with small amounts of serous fluid.

  • Pericardium: Membrane surrounding the heart; fluid referred to as pericardial fluid.

  • Pleural cavity: Space containing the lungs; fluid referred to as pleural fluid.

  • Peritoneum: Large membrane covering surfaces of:

    • Stomach.

    • Small and large intestines.

    • Liver.

    • Superior bladder and uterus.

• Normal cavities do not contain significant fluid; diseases can lead to fluid accumulation.

Build Up of Fluid

• Pericardial Effusion: Abnormal fluid accumulation around the heart.

• Pleural Effusion: Abnormal accumulation of fluid causing compressed lungs.

Cerebrospinal Fluid (CSF)

• Central nervous system (CNS) is protected by the skull and vertebrae and has three membranes:

  • Dura mater: Major protective layer for the brain and spinal cord.

  • Arachnoid mater: Thinner middle membrane.

  • Pia mater: Inner membrane that lies directly on the surface of the brain and spinal cord.

• CSF occupies the subarachnoid space (between the arachnoid and pia mater) and provides:

  • Protection and support for the CNS.

  • Circulation of nutrients and removal of waste.

• Normal total volume of CSF in adults: $90-150 ext{ mL}$.

Synovial Fluid

• Synovial fluid is a viscous fluid located between the junction of two or more bones (joints).

• Synovium: Membrane composed of mononuclear synovial cells that secrete synovial fluid into the joint space.

• Functions include:

  • Lubrication of joints.

  • Facilitating movement due to its viscosity, primarily from hyaluronic acid.

• Large joints may contain approximately $1 ext{ mL}$ of fluid.

Collection and Preparation

• Body fluid extraction is invasive and specimens are considered irretrievable; hence, quality is critically important.

• Collection process includes:

  • Performed only by physicians or properly trained personnel.

  • Fluids are aspirated using sterile needles and placed in tubes.

• Bodily fluids must be processed immediately as cells within these fluids rapidly lose viability, and degeneration can alter morphology.

Analysis and Clinical Correlations

• Automated body fluid analysis is feasible on modern analyzers but primarily gives WBC and RBC counts only; applicable only for serous fluid and some synovial fluid, not CSF.

• Manual cell counts through hemacytometer are the preferred method for fluid cell counts.

  • If specimen is clear, it is counted undiluted.

  • Both sides of the counting chamber are counted, with an agreement of ±10%.

Microscopic Examination

• Due to low cell concentrations, slides of body fluid are prepared through cytocentrifugation.

• Cytocentrifugation concentrates cells onto slides using specialized centrifuge-funnel apparatus:

  • Though preferred, cytocentrifugation can create artifacts that distort morphology.

  • Common artifacts include:

    • Irregular fragments.

    • Projections.

    • Vacuolization.

• Addition of albumin before centrifugation helps preserve cellular morphology.

• Slides must be prepared and examined even if no cells are observed under the hemacytometer.

Cellular Components

• Under normal conditions, body fluid cells are primarily composed of peripheral blood cells that migrate through endothelium, along with a small number of epithelial cells from membranes.

• Neutrophils: Frequently observed in serous and synovial fluids, typically comprise < 25% of total differential.

• Morphology: Mostly typical but may exhibit artifactual hypersegmentation, degeneration, and vacuolization.

• Lymphocytes: Vary in appearance, usually exhibiting reactive morphology, potentially with irregular shapes and cytoplasmic projections.

Tissue Specific Cells

• Macrophages: Transformed monocytes that may appear larger with abundant vacuoles.

• Monocytes may also be identified during analysis.

• Tissue cells are benign, occurring in all fluids, and should be differentiated from malignant cells.

• Benign mesothelial cells: Found in serous fluids, characterized as large cells with moderate to abundant, light to dark blue cytoplasm and an eccentric nucleus with a smooth outline and fine chromatin pattern.

  • May appear in sheets; distinct cytoplasmic boundaries help differentiate from malignancy.

• CSF and synovial fluid may contain tissue cells but are rare.

Serous Analysis

• Effusion: Abnormal fluid collection in a membrane.

  • Transudate: Effusion resulting from systemic disease.

  • Exudate: Effusion due to a primary disease of the compartment (e.g., lung).

  • Typically presents as cloudy, turbid, or purulent due to lipids or WBC count.

  • Chylous effusion: Exudate resulting from lymphatic obstruction or leakage.

  • Pseudochylous effusion: Persistent effusion due to conditions like tuberculosis or rheumatoid arthritis-related inflammation (not lymphatic-related).

Cellular Response

• The cellular population in serous fluid can change in response to pathological states similarly to peripheral blood.

• Neutrophils: Count increases during bacterial infections; bacteria and fungi may also be identified during infections.

• Malignant cells typically appear as a homogeneous population characterized by:

  • Large size with multilayered formations.

  • Irregular nuclear membrane and multinucleation.

  • Bizarre vacuolization and abnormal inclusions.

  • Nuclear molding: The nucleus molds around the shape of other cells.

Pleural Effusions

• Reminder: Effusion is an abnormal accumulation of fluid in a cavity.

• Occurs when fluid production exceeds absorption and can be bilateral or unilateral based on underlying causes.

• Excess fluid may restrict lung expansion, leading to dyspnea and mild hypoxemia.

• Removal of fluid is performed via thoracentesis before testing (EDTA for hematology).

• Gross appearance can provide diagnostic clues, such as:

  • Bloody fluid without trauma generally indicates malignancy.

  • Empyema: Presence of pus suggests bacterial pneumonia or a ruptured abscess.

Pericardial Effusion

• Pericardial Effusion is characterized by abnormal fluid accumulation in the pericardial space, usually from damage to the lining.

• The effect on cardiac function is determined by fluid volume, rate of formation, and elasticity of the pericardial membrane.

• Severe pressure from fluid can restrict the heart, leading to critical dysfunction or death.

• Removal of fluid, termed pericardiocentesis, prepares the sample similarly to thoracentesis.

Peritoneal Effusions

• Peritoneal effusion can also be referred to as ascitic or paracentesis fluid, associated with similar causes of effusions.

• Caused by conditions like alcoholic cirrhosis.

• The procedure for fluid removal is known as paracentesis and may include a flush with normal saline (lavage).

• Patients in renal failure may undergo continuous ambulatory peritoneal dialysis (CAPD), where dialyzing fluid is instilled into the cavity to remove bloodstream impurities.

Table 30-5: Body Fluid and Collection Procedure Nomenclature

• Fluid Name | Collection Procedure | Body Cavity/Region

• Pleural (serous) | Thoracentesis | Pleural cavity/lungs

• Pericardial (serous) | Pericardiocentesis | Pericardial cavity/heart

• Peritoneal, ascites (serous) | Paracentesis | Peritoneal cavity/abdomen

• Cerebrospinal | Lumbar puncture/spinal tap | CNS; subarachnoid space/brain and spinal cord

• Synovial | Arthrocentesis | Synovial cavity/joints

Laboratory Analysis

• EDTA in specimen tubes helps preserve cell morphology and prevent clotting for bloody specimens.

• If clotted, morphology examination is performed, noting inaccuracies in cell count.

• Normal serous fluid appears pale yellow to straw-colored and transparent.

• Grossly bloody and turbid specimens indicate underlying disease.

• Both automated analyzers and manual cell counts are performed to determine cellular concentration.

• Cloudy or turbid specimens may be diluted before manual counting.

• Cytocentrifuged slide examination is conducted regardless of initial counts.

Microscopic Analysis

• Circular field of stained elements should be scanned at low power for identifying cellular clumps or large cells indicating malignancy.

• Standard differential counts on high power using oil immersion, including macrophages and mesothelial cells categories.

• If fewer than 100 cells are noted, all cells are counted with a percentage provided.

• Small numbers of mesothelial cells slough into serous cavities during inflammatory conditions, proliferating and shedding more abundantly.

• Reactive changes in mesothelial cells can pose challenges for correct identification, especially if clustered due to centrifugation.

CSF Collection

• CSF collection occurs with a lumbar puncture, ensuring aseptic conditions and avoiding trauma.

• Approximately $2-4 ext{ mL}$ is collected in 3-5 sequential non-additive tubes.

• Tubes should be filled in the following sequence:

  • Tube 1 for chemistry tests (usually contaminated with peripheral blood).

  • Tube 2 for microbiology (less affected by contamination).

  • Tube 3 for cell count and differential (least affected by bleeding during the tap).

  • Additional tubes for specialty tests as necessary.

CSF Analysis

• Gross inspection identifies color, clarity, and viscosity: should resemble clear water.

• Verify tube numbers.

• Bloody specimens may indicate intracranial hemorrhage or a traumatic tap.

  • Traumatic taps initially show high blood but decrease in sequence, while hemorrhagic specimens maintain bloodiness throughout collection.

• Xanthochromia: Yellow to light orange discoloration in CSF after centrifugation, typically signifying bilirubin presence from SAH or jaundice.

CSF Microscopic

• CSF counts must be conducted manually, with normal CSF being nearly devoid of cells.

• In a normal patient, any observed WBCs tend to be lymphocytes.

• In traumatic taps, cell counts reflect peripheral blood populations, with approx. $1-2 ext{ WBCs per 1000 RBCs}$.

• Neutrophil counts rarely naturally occur in CSF; high counts indicate disease.

• Reactive lymphocytosis may occur in instances of viral meningoencephalitis or post-chemotherapy.

CSF Microscopic Observations

• Increased monocytes may indicate a mixed reaction.

• Erythrophagocytosis can appear in CNS hemorrhages.

• In patients with previous hemorrhages, degraded hemoglobin can show up as hemosiderin in macrophages (dark brown or black granules).

• Leukemic cells may manifest in CSF, particularly late in disease progression, which is a negative prognostic sign.

• Microorganisms such as fungi and bacteria may appear both intracellularly and extracellularly; PCR is utilized to detect viruses.

Table 30-6: Normal Values for Cerebrospinal Fluid

• Appearance: Clear, colorless, watery

• RBC Count: <$1/ ext{μL}$

• WBC Count: $0-5/ ext{μL}$ (adults); $0-30/ ext{μL}$ (infants up to 1 year)

• WBC Differential:

  • 3% neutrophils

  • Ratio of lymphocytes to monocytes/macrophages is approximately $70:30$.

• Rare ependymal cells may also be present.

Synovial Fluid

• Analysis yields insights into inflammatory and degenerative joint diseases.

• Effusion: Increased volume categorized into inflammatory, non-inflammatory, septic, or hemorrhagic origins.

• Frequently analyzed when septic arthritis or crystal-associated joint diseases such as gout are suspected.

• Collection procedure referred to as arthrocentesis; more common in children for septic arthritis concerns.

Synovial Analysis

• Routine analysis includes:

  • Crystal examination

  • Cell count

  • Differential

  • Chemistry and microbiological testing

• Normal synovial fluid should be:

  • Transparent

  • Colorless to straw-colored

  • Viscous and non-clotting

• Hemarthrosis: Bloody aspirate results may reflect either hemarthrosis from joint bleeding or needle trauma.

• Septic arthritis manifests as purulent fluid with the presence of bacteria and pus.

Synovial Microscopic Analysis

• Effusions classified by volume and leukocyte types present.

• Crystal-induced diseases characterized by the presence of crystals.

• Cell counts performed on hemocytometer.

• To reduce viscosity, samples may require pretreatment with hyaluronidase.

• Differential counts classify the type of effusion, performed on cytocentrifuged Wright-Giemsa stained samples showing primarily lymphocytes, macrophages, and lining cells; neutrophils should not exceed 25%.

Crystal Analysis

• Every synovial fluid sent for a cell count is examined for crystals.

• Crystal analysis can be conducted on “wet prep” and unstained cytocentrifuged slides.

• Polarized light microscopy is utilized to detect and distinguish diagnostic crystals:

  • Crystals exhibit birefringence (refract light), requiring a rotating filter (polarizer) beneath the slide stage and a filter (compensator) between the objectives and oculars.

  • This setup yields a dark field, enhancing birefringent visualization of materials.

Synovial Crystals

• Common crystals found in synovial fluid include:

  • Monosodium urate (MSU): Characteristic of gout, presenting as long, thin, needle-like crystals, either intracellularly or extracellularly, and are strongly birefringent (yellow when parallel, blue when perpendicular).

  • Calcium pyrophosphate dihydrate (CPPD): Present during CPPD deposition disease (pseudogout); appears as short rods or rhomboids and is weakly birefringent (opposite to MSU).

Gout vs Pseudogout

• Visual identification can differentiate between gout and pseudogout based on the characteristics of the crystals observed under polarized light.

Summary

• All body fluids should be processed immediately, examined for volume, color, clarity, and clot presence.

• Hematology body fluid tests encompass:

  • Total cell count

  • Morphological and differential analysis

  • Crystal analysis (specific to synovial fluid)

• CSF collected in sterile, non-additive tubes, filled sequentially.

• Body fluid slides are prepared through cytocentrifugation for differential assessments and malignant cell examinations, understanding that cytocentrifugation may introduce artifacts.

• The most common crystals identified in synovial fluid are MSU and CPPD.