Automating Microscope Based Particle Counting – USP 788 Particulate Matter in Injections

Optical microscopes are commonly used for particle counting to quantify the level of foreign particulate matter in orally inhaled, nasal drug products and injection liquids. With the addition of a high-resolution digital camera, PAX-it! image-analysis software and a motorised microscope XY scanning stage, this process can be semi-automated or even fully automated, significantly increasing throughput, accuracy and repeatability.

Counting these particles is an enormously important aspect of product production. The most common method, microscopic particle counting is outlined as one of the guidelines in USP 788 (Particulate Matter in Injections). Click here for the microscope guidelines on USP 788.

Manual Microscope-Based Particle Counting has been used for decades however it is time consuming, tedious and subject to human error. Automating the entire workflow results in enormous savings in time and great gains in repeatability and precision. Outlined below are some of the advantages for automating this process.

PAX-it, Mason Technology supplying a comprehensive 21 CFR Part 11 compliant imaging and security solution for your microscope.

Mason Automated Semi Automated System

 

Figure 1. Automatic particle detection software. All particles can be counted, measured and classified for further evaluation.

PAX-it! Image Analysis tools can use density, color, shape factors, and size filters to detect and sort objects or areas within your images. Filters may be applied to disregard objects of certain shapes or sizes, or to split the results into bins related to specific measurements. Touching particles which manually could have been counted as one can also be auto-separated in the software giving repeatability on results and taking out human error all with the click of a button. While semi-automated counting relies on the operator manually positioning the XY stage underneath the microscope and then manually triggering image capture at various fields of view, PAX-it! integrated motorised stage module can automate the entire workflow, resulting in enormous savings in time and great gains in repeatability and precision

Fully automated systems offer additional benefits as all relevant data including spreadsheets, histograms and reports can be generated in compliance with any company-defined or other predetermined standard (such as USP 788). Data can be archived into an integrated database automatically, virtually eliminating the potential for data loss that can occur when operators forget to input data. Administrators can customise and define database fields according to their specific experiment requirements and workflow. Later, users can search by any of these fields (such as batch number, operator name, customer name, date etc.) to find important information or to look for patterns. In addition PAX-it! software is designed to comply with 21 CFR Part 11 guidelines concerning electronic records and electronic signatures.

Fully automated systems help streamline the analysis, production and verification of manufacturing procedures and standards for these types of pharmaceutical products. For more information on this procedure please contact us.
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A suitable quantity of powder is examined (for example, 10 to 100 mg), and suspended in 10 mL of a suitable medium in which the powder does not dissolve, adding, if necessary, a wetting agent. A homogeneous suspension of particles can be maintained by suspending the particles in a medium of similar or matching density and by providing adequate agitation. A portion of the homogeneous suspension is placed on suitable counting cell, and an area corresponding to not less than 10 µg of the powder is examined under the microscope. All the particles that have a maximum dimension greater than the prescribed size limit are counted. The size limit and the permitted number of particles exceeding the limit are defined for each substance.

The counting of particles can sometimes be tedious and also subject to human error. By using the PAX-it automated particle detection software a threshold can be applied in which it only counts particles of a certain size giving repeatability. For a more detailed description on automating particle counting please see “Automating Microscope Based Particle Counting” in applications.

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The measurement of particle size varies in complexity depending on the shape of the particle, and the number of particles characterised must be sufficient to ensure an acceptable level of uncertainty in the measured parameters. Additional information on particle size measurement, sample size, and data analysis is available, for example, in ISO 9276. For spherical particles, size is defined by the diameter. For irregular particles, a variety of definitions of particle size exist. In general, for irregularly shaped particles, characterisation of particle size must also include information on the type of diameter measured as well as information on particle shape. Several commonly used measurements of particle size are defined below (see Figure 1):

Mason commonly used measurements of particle size

Figure 1. Commonly used measurements of particle size.

Gram staining methods include the four-step method: crystal violet (primary stain), Iodine (mordant), alcohol or alcohol–acetone (decolorizer), and safranin (counterstain). In the three-step method, the decolorization and counter staining steps are combined. Under optimal conditions, Gram-positive organisms retain the crystal violet stain and appear blue violet. Gram-negative organisms lose the crystal violet stain, so they contain only the counterstain safranin and appear red. Some bacteria may be Gram-variable. Common pitfalls in this method are that heat fixation may cause Gram-positive cells to stain Gram-negative, and older cultures may give Gram-variable reactions. Using too much decolorizer could result in a false Gram-negative result, and not using enough decolorizer may yield a false Gram-positive result. One variation that has advantages in some situations is to perform a methanol, rather than heat, fixation of the bacterial smear. In some cases alcohol fixation may give more consistent Gram stain results. In either method a Gram-positive and a Gram-negative control should be included to allow identification of errors in staining. The Gram-staining reaction must be read under a microscope at 100X high power and cellular morphology can be simultaneously ascertained from this.

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Companies who rely on PAX-it!:

Merck, PAX-it - Trusted by Companies Around the World
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Pfizer, PAX-it - Trusted by Companies Around the World
Medtronic, PAX-it - Trusted by Companies Around the World
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Centers for Disease Control and Prevention
Lucent Technologies, PAX-it - Trusted by Companies Around the World
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Texaco, PAX-it - Trusted by Companies Around the World
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Procter and Gamble, PAX-it - Trusted by Companies Around the World
Dupont, PAX-it - Trusted by Companies Around the World

As the principal distributor for PAX-it! in the UK and Irish market, Mason Technology can advise and install the compliant software in your business. Get in touch with one of our team for further information on how PAX-it! can benefit your organisation.


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