Reliability and Accuracy of Intradermal Injection by Mantoux Technique, Hypodermic Needle Adapter and Hollow Microneedle in

Pigs

James J Norman†, Jyoti Gupta†, Samir R Patel, Sara Park, Mark R Prausnitz*

All authors:Georgia Institute of TechnologySchool of Chemical and Biomolecular Engineering311 Ferst DriveAtlanta, GA 30332

† Co-first authors

* Corresponding author:Mark R Prausnitzprausnitz@gatech.eduP: 404-894-5135F: 404-894-2291

AbstractWe compared the ability of three intradermal delivery devices to administer an intended dose to pig skin in vivo and target that dose to the dermal rather than subcutaneous layers. The three devices were: a standard hypodermic needle and syringe for the Mantoux technique, an adapter designed to facilitate proper hypodermic needle and syringe use and a hollow microneedle. Reliability was determined as the percentage of the administered dose that entered the skin, as opposed to remaining in the device or on the skin surface. The intradermal adapter (97.6% ± 1.5% delivered, mean ± standard deviation), Mantoux technique (95.4% ± 4.9%) and hollow microneedle (94.9% ± 0.3%) exhibited similar reliability. Accuracy was determined as the percentage of the dose that entered the skin that localized in the dermis. All three devices achieved similar accuracy: hollow microneedle (99% ± 12% delivered to the dermis, median ± standard deviation), intradermal adapter (97% ± 16%) and Mantoux technique (92% ± 21%). We conclude that intradermal injection by all three methods studied provided reliable delivery to the skin and provided accurate localization of delivery within the dermis. Next-generation designs of these devices have now received clearance from the FDA and are used as medical products and/or in clinical trials.

KeywordsIntradermal injection, microneedle, Mantoux technique, hypodermic needle adapter, skin histology

IntroductionIntradermal injections are used for vaccination against tuberculosis, rabies and influenza [1], diagnostic tests for allergies or tuberculin sensitivity [2], local dermal anesthesia [3], and other dermatological procedures. Recent research has also shown advantages of intradermal delivery for drugs typically given subcutaneously or intramuscularly. Examples include insulin [4, 5] and parathyroid hormone [6], where rapid drug uptake in the skin provides improved pharmocokinetics, and several vaccines for which skin-resident immune cells contribute to a stronger immune response [7].

The standard method for intradermal injection is the Mantoux technique using a hypodermic needle and syringe [8]. The needle for intradermal delivery is typically finer in gauge and shorter in length than needles for other types of injections, e.g., 1/2 inch, 28 gauge needles for intradermal injection compared to 1 inch, 24 gauge needles for a typical intramuscular injection. For the Mantoux technique, the needle is inserted at a grazing angle to skin, i.e., 10-15° compared to 45° for subcutaneous and perpendicular for intramuscular injections. This method is reliable in the hands of a skilled administrator and requires no tools beyond a needle and syringe. However, this method is difficult to learn [9] and not sufficiently robust to variables like needle gauge [10]. For intradermal delivery to become more widespread for vaccination or home delivery of drugs, a simpler delivery method is needed.

Several alternatives to the Mantoux technique exist. This study compares the reliability and accuracy of three intradermal delivery devices: a standard hypodermic needle and syringe for the Mantoux method, an adapter designed to facilitate proper hypodermic needle and syringe use, and a hollow microneedle measuring less than 1 mm in length. This study used early-generation, experimental devices during their development. After this study was completed, next-generation versions of the intradermal adapter and the hollow microneedle have been developed and achieved FDA clearance.

We believe this is the first study to made direct comparisons between different intradermal delivery methods and devices to assess their reliability and accuracy, although skin delivery tests have been published for microneedles in isolation [11-13]. Specifically, this study compares the ability of each device to deliver the intended dose to tissue (“reliability”) and to target drug in the dermal layers rather than the subcutaneous space (“accuracy”).

MethodsThe Devices The three intradermal delivery devices used in this study and shown in Fig. 1 are: a standard hypodermic needle and syringe for the Mantoux technique, a hypodermic needle and syringe with a plastic adapter that facilitates proper intradermal insertion into skin, and a hollow microneedle. Mantoux and adapter injections used a ½ inch, 28 gauge needle mounted on a 1 mL syringe (Fig. 1a). The adapter was a second-generation prototype provided by PATH (Seattle, WA) and developed in collaboration with SID Technologies (Newton, PA); it has a flat plastic base that resembles a butterfly needle and fixes the angle at which the needle meets the skin (Fig. 1b). Mantoux injections were administered bevel-up according to standard protocol [8], whereas adapter injections were administered bevel down since this was found to be more effective in pilot studies. Borosilicate glass microneedles, 850 µm long, were manufactured by our group at Georgia Tech (Fig. 1c) and inserted using rotary device described earlier [14]. The investigator carrying out the injections in this study received on-site training on the proper use of the intradermal adapter and the hollow microneedle from the manufacturer of these devices. The same investigator gave all injections using all three devices in this study.

Reliability MeasurementFive male feeder pigs were used for this study, each 20-40 kg. The pigs were placed under anesthesia and had their back hair trimmed using clippers. We chose sites for injection that were 1.5-2.5 mm in skin thickness with the aid of a DermaScan C ultrasound scanner (Cortex Technology, Denmark). Sites were

wiped with alcohol prior to injection. One hundred microliters of sulforhodamine B at a concentration of 10-3 M in phosphate buffered saline (PBS) was loaded into each device and injected. After each injection, the skin surface was wiped with a pre-wetted gauze pad and tape-stripped to remove any dye present on the skin surface. Sulforhodamine was extracted from the gauze and tape in 4 mL PBS. The devices were also rinsed in 4 mL PBS to extract remaining dye. Collected samples were analyzed using a spectrophotometer (SpectraMax Gemini, Molecular Devices, Sunnyvale, CA). In this way, we directly determined the amount of dye on the skin and in the device, and determined the amount of dye in the skin by a mass balance calculation. The investigator performing the administrations also made qualitative observations about the usability of the experimental devices. All animal procedures were approved by the Georgia Tech IACUC.

Accuracy MeasurementThe same injection procedures were used in five freshly euthanized pigs to deliver 0.4% w/v trypan blue dye. Immediately after injection, we took 8-12 mm biopsies of the skin and fat. Samples were frozen on dry ice in optimal cutting temperature solution. An independent, blinded histologist cut samples into 10 µm sections using a microtome (Microm HM 550, Microm, Waldorf, Germany). Sets of eight sections 500 µm apart were taken from tissue where dye was visible. Digital pictures of the sections were acquired under similar lighting conditions. A separate blinded investigator used Adobe Photoshop to determine the distribution of dye in the dermal layers compared to the fat layer by calculating the relative proportion of blue pixels in the two layers.

ResultsReliability: Delivery of Intended DoseWe delivered a fluorescent dye to pig skin and determined the fraction of the intended dose that was delivered to the skin (or subcutaneous tissue) rather than remaining on the skin surface (i.e., a “wet” injection) or within the device. Fig. 2 shows the reliability results, where the intradermal adapter (97.6% ± 1.5% delivered, mean ± standard deviation), Mantoux technique (95.4% ± 4.9%) and hollow microneedle (94.9% ± 0.3%) performed similarly with reliability results that were indistinguishable from each other (Tukey-Kramer test following ANOVA: all p > 0.05). Accuracy: Targeting Dose to the DermisWe delivered a staining dye to pig skin to determine how accurately devices targeted drug to the dermis. Fig. 3 shows characteristic histology images for each device. Intradermal injection using the Mantoux technique and the intradermal adapter produced similar dye distributions in the skin. Typically, the injection appeared throughout the thickness of the dermis, with more intense dye staining in the center (presumably marking the site of injection from the needle orifice) and a small amount of dye appearing subcutaneously. The hollow microneedle injection profile appeared notably different, where intense dye staining appeared in the superficial dermis just below the epidermis and the overall distribution of the injection was more superficial than injection with a hypodermic needle. This observation is presumably due to the shallower insertion depth of the microneedle.Fig. 4 quantifies the accuracy of injection within the dermis. The hollow microneedles (99% ± 12% delivered to the dermis, median ± standard deviation), intradermal adapter (97% ± 16%), and Mantoux technique (92% ± 21%) all performed similarly with accuracy results that were indistinguishable from each other (Kruskal-Wallis test, p = 0.23). Considering the consistency of intradermal targeting, each method showed most injections localized mostly to the dermis, with a few notable outliers. Statistical analysis showed that the microneedles had significantly lower variability in dermal targeting compared to intradermal adapter injections (F-test, p < 0.05).

Qualitative Observations on Device UsabilityThe investigator performing the injections made subjective observations on the usability of each device. Because the investigator was an expert at intradermal injections, she found the usability of the Mantoux method to be good. However, broader clinical experience shows that healthcare professions have

difficulty with the Mantoux technique if they lack recent training or frequent injection practice [15, 16]. Because the investigator was expert at intradermal injections without an adapter, she found using the intradermal adapter to reduce usability compared to the Mantoux technique, because it required extra steps and got in the way. However, for those who are not expert in giving intradermal injections by the Mantoux technique, the investigator believed that the adapter would increase usability by reducing the skill level required for intradermal injection. A next-generation version of this prototype adapter is now available from West Pharmaceutical Services (Exton, PA).

Concerning the microneedle, the investigator noted that no special skill was needed to insert the microneedle into the dermis, but the delivery procedure was slower than other methods and required a higher pressure. In addition, the microneedle device used in this study was an early research prototype, where the microneedle was made of glass and the holder/insertion device was relatively large and cumbersome. The use of a modified conventional syringe with a larger-bore microneedle manufactured by Becton Dickinson (Franklin Lakes, NJ) or an array of multiple hollow microneedles made by NanoPass Technologies (Nes Ziona, Israel) has largely overcome these usability limitations of the early prototype studied here.

DiscussionThree intradermal delivery devices were compared for reliability, i.e., their ability to deliver an intended dose to tissue, and accuracy, i.e., their ability to target that dose to the dermis. The devices tested were a standard hypodermic needle and syringe for the Mantoux technique, an intradermal adapter attached to a hypodermic needle and syringe, and a hollow microneedle. We believe this is the first study to directly compare the performance of a variety of intradermal delivery devices and the first published study to include the intradermal adapter.

In terms of reliability, all three performed similarly well. The two hypodermic needle methods and the microneedle method all injected over 94% of the intended dose on average, with little variability. The three devices showed no significant difference in the median accuracy of delivery localized to the dermis, which ranged from 92% for the Mantoux technique to 99% for the hollow microneedle. The hypodermic needles likely performed well at dermal targeting because the Mantoux technique and the intradermal adapter are designed to keep the needle orifice within the dermis. The microneedle likely performed well because the needle itself is too short to penetrate past the dermis.

There were differences in the consistency (i.e., as measured by standard deviation of the measurements) of accurate delivery to the dermis. Hollow microneedles more consistently targeted the dermis compared to the intradermal adapter. One possible explanation is that microneedles were designed intentionally for intradermal delivery whereas hypodermic needles were adapted for the task. A related observation is that hollow microneedles typically deposited dye closer to the skin surface compared to hypodermic needles (see Fig. 3). This observed uniformity in targeting is important for drug delivery, since it is easier to control for a small fraction of a dose regularly missing the dermis compared to a small fraction of injections sporadically missing the tissue altogether.

The primary limitations of this study involved using a single investigator for administration and using dye delivery in pigs as an approximation for drug delivery in humans. As we mentioned previously, the devices were not engineered or optimized for delivery in pig skin, and this may have affected our results.

ConclusionA standard needle and syringe, an intradermal adapter, and a hollow microneedle all provided good reliability of delivering the intended dose into the skin (95% - 98% reliable) and good accuracy of targeting the dermis (i.e., 89% - 99% accurate). The hollow microneedle delivered the injection primarily in the superficial dermis, whereas the two hypodermic needle techniques delivered the injection

throughout the dermis. In addition to these performance characteristics, decision makers should consider usability, cost, and administrator training when choosing an intradermal delivery device.

AcknowledgementsDonna Bondy provided administrative assistance. We thank PATH (Seattle, WA) for providing devices for this study. Brian Bondy assisted with histological analysis. This work was supported by a grant from PATH.

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Figure Legends

Figure 1. Intradermal delivery devices used in this study. (A) A standard hypodermic needle and syringe for the Mantoux technique. (B) A needle and syringe with a plastic adapter that facilitates proper insertion into skin. The inset shows the portion of the needle that extends outside the adapter. (C) A hollow microneedle in a stainless steel holder. Arrows show the site of the needle in each device.

Figure 2. Reliability of the intradermal delivery devices to deliver an intended dose to pig skin in vivo. Data points represent average ± standard deviation, n=10-20).

Figure 3.Characteristic histological sections for each injection method. (A) Mantoux method, (B) intradermal adapter, (C) hollow microneedle.

Figure 4. Accuracy of the intradermal delivery devices to target drugs to the dermis rather than subcutaneous space. Each point on the scatter plot represents a separate injection. The dashed line represents the median for each device.

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