Synovial fluid control

Details Synovial fluid control Synovial fluid control

2001-08-23

2003-11-25

Tate, Christopher R. (Department: 1651)

Optics: measuring and testing

By polarized light examination

With birefringent element

C359S494010, C435S007240, C435S007250

Reexamination Certificate

active

06654120

ABSTRACT:

BACKGROUND
This invention relates to the measurement of cell counts and identification of crystals in synovial fluid samples and more particularly in providing a control reference material for the quality control of the measurements of these cells and the identification of these crystals of interest.
Synovial fluid, often referred to as joint fluid, is a viscous liquid found in joint cavities and supplies nutrients to the cartilages and acts as a lubricant to the surfaces of the frequently moving joints. It is formed as an ultrafiltrate of the plasma across the synovial membranes, into which a mucopolysaccharide containing hyaluronic acid and a small amount of protein is secreted by the cells of the synovial membrane. The chemical composition of the synovial fluid is essentially the same as human plasma except for the high molecular weight proteins which are normally absent in the synovial fluid. It is established that total cell count, differential white blood cell count and crystal identification on synovial fluid can provide valuable information regarding infection, inflammation, and irritation of the joint spaces. Total cell counts are usually performed in a counting chamber, while crystals are analyzed using light and polarized light microscopy. Red blood cells are seen in synovial fluid samples either as a result of a traumatic tap of the joint or as a result of a hemorrhagic condition. Lymphocytes and other white blood cells are seen during conditions of nonseptic inflammation while neutrophils are seen during conditions of bacterial sepsis and crystal induced inflammation. Identifying the causative crystals in the synovial fluid, herein also referred to simply as fluid, especially if they are found intracellularly in neutrophils and macrophages, is pathognomic for a crystal induced arthritis. Pathognomic means characteristic of a disease condition or stage.
Gout and pseudogout comprise the two major crystal induced arthritides. The presence of intracellular monosodium urate (MSU, hereinafter) crystals in neutrophils and macrophages is found in 90% of patients having an acute attack of gout. These crystals, however, are also seen in synovial fluid between attacks in 75% of patients, suggesting that multiple factors contribute to an acute episode. Pseudogout, or calcium pyrophosphate deposition disease, is characterized by the presence of calcium salts in cartilage and calcium pyrophosphate dihydrate (CPPD, hereinafter) crystals in synovial fluid. Unlike gout, no single serum metabolite is responsible for the disease. Hereditary metabolic problems or endocrine disorders, such as hypothyroidism and hyperparathyroidism, that elevate calcium levels in blood can lead to pseudogout. More commonly, pseudogout is associated with degenerative arthritis, demonstrated by X-ray evidence of articular cartilage calcification. As many as 50% of adults may have CPPD deposits in their joints at the time of their deaths. Techniques for crystal analysis focus on MSU and CPPD, the two most common crystals. MSU crystals are needle shaped and produce negative birefringence with polarized light. CPPD are often rhomboid, but are also seen as needles and rods, and are weakly birefringent with polarized light, producing positive birefringence. The analyses of these crystals using polarized microscopy and the counting of the cells using counting chambers are known. Other birefringent materials that may also be found in the synovial fluid include calcium oxalate, cartilage fibers, collagen, cholesterol, hydroxyapatite, betamethasone acetate, cortisone acetate, methyl prednisone acetate, prednisone tebulate, triamcinolone acetonide, triamcinolone hexacetonide, EDTA (ethylenediamine tetraacetate), fat or cholesterol esters, lithium heparin, starch granules and debris.
Analysis of synovial fluid is widely accepted as a crucial aid in the evaluation of joint diseases. Identification of crystals in joint fluid establishes a definite diagnosis of these diseases and the leukocyte count can be used to broadly group diseases as inflammatory or noninflammatory. Although these analyses are diagnostically crucial, there is no established available reference control material designed to monitor the accuracy, reliability and reproducibility of these tests or analyses. Incidents of misclassification between inflammatory and noninflammatory due to a wide range of cell count reported are known and discrepancies in crystal identification causing false positives and false negatives have been reported. These affect both diagnosis and treatment. Several factors have been proposed as reasons for these errors such as lack of training and experience with the test methodologies by the laboratory analysts, the infrequency of testing performed in synovial fluids, the differences in the quality and nature of the equipment used for performing the tests, the difference in the diluting fluids used for the specimens, the amount of crystals present in a sample or specimen, differences in the viscosity of the samples, procedural variations in the handling and pipetting of the samples, and differences in time after which a sample or specimen is tested after collection. Clearly, quality control measures are needed for these tests.
It is therefore an object of this invention to provide a reference control material that can be easily prepared in commercial quantity to help improve the reliability of the test results obtained by testing laboratories.
It is also an object of this invention to provide a pathognomic reference control material having red and or white blood cells and or crystals for identifying and differentiating patients with degenerative arthritis from those patients suffering from gout.
It is a further object of this invention to provide several diluent or base materials suitable for suspending cell/s and/or crystals for the reference control material.
SUMMARY OF THE INVENTION
Laboratories have used synovial fluid obtained from the knee of patients with relatively inactive rheumatoid arthritis as a negative control in the analyses of patients' synovial fluids. Others have spiked human synovial fluid with known amounts or either monosodium urate (MSU) or calcium pyrophosphate (CPPD) crystals or a combination of these. However, to use human synovial fluid as a base for the preparation of a reference control material is not practical because of its limited quantity. Further, simply spiking the synovial fluid from a normal individual with the crystals or other birefringent materials of interest do not present a pathognomic reference control material because white blood cells and/or red blood cells may not be present in these spiked samples. This invention offers several synovial fluid reference control materials interchangeably referred to herein as synovial fluid control materials of broad flexibility, variety and usage. For a pathognomic synovial fluid reference control material, hereinafter referred to as pathognomic control, it is most preferred to spike a simulated synovial fluid base. This pathognomic control comprises a desired amount of fixed red blood cells, the desired amount less than 5000 cells/ul (microliter), preferably 30-300 cells/ul; a desired amount of fixed white blood cells, the desired amount less than 10000 cells/ul, preferably 300-1500 cells/ul; a desired amount of calcium pyrophosphate crystals, the desired amount less than 300 mg/l (liter), preferably 150 mg/l; and/or a desired amount of sodium urate crystals, the desired amount less than 300 mg/l (liter), preferably 80 mg/l. Because it is a pathognomic control, not all of the components mentioned above need be present in a single formulation but only those required or present in a particular disease state of interest. A simulated synovial fluid base comprises glucose, sodium lactate, potassium and sodium chloride and a preservative. The simulated synovial fluid base preferably contains hyaluronic acid and most preferably contain human serum albumin. The level of cell counts and crystals may vary based on the apparatus and test procedure used

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