A7   In    addition    to   the    analytical    method    validation stipulated   in    the    bioanalytical guideline/guidance in each regulatory region, the following points should be considered when analysing samples derived from microsampling:

For liquid sampling, the following issues should be considered  1) ensuring sample homogeneity, for example by pipetting;  2) small volume handling issues (e.g. potential freezing/drying effects during the storage and subsequent freeze/thaw process, as applicable); 3) potential increase in the LLOQ  due  to  the  limited  sample  volume;  4)  impact  of  addition  of  anticoagulants  to  small containers/capillaries, resulting in dilution of the sample; 5) potential for increased adsorption of the analyte to the collection container (i.e. increased surface area to volume ratio); 6) keeping the appropriate preservation  conditions  of the  sample;  7) risk of contamination  and difficulty of repeated sampling using some methods.

For  dried  sampling  techniques  (e.g.  spotting  onto cellulosic or  non-cellulosic card,  polymer matrix, etc.), it is important to select the method with adequate and reproducible recovery and minimal matrix interference with detection of the drugs.   If the sub-punch of the dried spot approach is used, it is important to ensure that detection of the analyte is not affected by different haematocrit values, especially for small molecule drugs. The effect of haematocrit on analyte detection can be measured by using blood with different haematocrit values and spiking with test drugs of known concentrations.   It is  also important to confirm the uniformity of the spots by evaluating analyte levels from multiple samples punched out from one spot or, by evaluation, for example, using radiolabels.   Both  of these  issues can be minimized if an accurate volume of blood is collected on the device and the entire sample is subsequently analysed.

Incurred sample   reanalysis    (ISR)    should    be    conducted    according   to    each    regional guidance/guideline, if described.   When  doing  ISR,  care  should be taken to ensure sufficient sample volumes or numbers of replicates (e.g. spots, containers, or tips) are retained for ISR.

A6 When blood sampling is performed on the main study animals, it is important to consider the effect of blood collection on the physiological condition of the  animals.   The main  factors to consider include;  1)  the  volume  and  the number of samples taken in  a given period; 2) the properties of  the test   drug (e.g. its effects on red blood cells, anticoagulant effect, or haemodynamic  properties);    3)  the  test  system (e.g. species, age, body weight, total blood volume); and 4) the site of collection.   As  frequent  and repeated blood  collection may affect physiological   data,    such   as    haematological   parameters,    sampling   protocols    should   be appropriately established even with microsampling.   It is prudent to record the relevant animal data, such as changes in body weight, food consumption, haematological parameters (e.g. red blood cell count, haemoglobin level, haematocrit value, mean corpuscular volume, electrolytes, total proteins), and any effect on the blood collection site (e.g. tissue damage, inflammation). Evaluation of these parameters compared to matching control animals, which have had the same number and volume of samples drawn as the test drug groups, will be important to   establish whether any suspected effects are related to test drug or to procedures, within the context of the specific   study   conditions.      If previous   studies   show   that   test   drug-related   changes   to haematological parameters could be exacerbated by frequent blood sampling, or it is suspected that the pharmacological action of the test drug may induce such effects, the use of satellite groups of animals for TK assessment would be warranted, even if microsampling techniques are used.  Alternatively,   sparse sampling* can  be used   in conjunction  with microsampling,   if scientifically justified.

*Sparse  sampling  in  TK  studies  usually  involves the collection of  a  few blood samples at specified timepoints from each animal in a treatment group. The samples are allocated at different time points for different animals, often allowing some replicates, and statistical inferences are then made about the concentration-time behaviour of the test compound. By using proper study designs, investigators can limit the number of samples and the amount of blood drawn, so as not to affect the animals' health status, yet still achieve the customary toxicokinetic objectives in a study.

A5 Blood can be collected from the tail vein, saphenous vein, etc., using capillary tubes or any appropriate miniaturized collection devices. The collected blood, and its derived plasma or serum, can be used to measure drug concentrations in either liquid or dried form.   In some cases, liquid samples can be diluted with the appropriate vehicles or blank matrices prior to storage, shipment and subsequent analysis.  Dried sample methods are also available, wherein the sample is usually applied directly onto cellulose-based or other types of materials and then dried. A fixed diameter sub-punch or the whole quantity of the sample on the card/device can be extracted and analysed. Recent and on-going advancements in microsampling methods have demonstrated the ability to collect precise volumes of blood, such that the entire sample can be used for analysis without additional volumetric measurements.   In  addition,  newly  developed  techniques  could  also  be considered with adequate validation.

A4 As with other approaches to kinetic sampling, in order to adopt a microsampling technique appropriately, a bioanalytical method should be developed and qualified (or validated for Good Laboratory Practice studies, in accordance with regulatory guideline/guidance in each region) to ensure the reliability of analytical results.   Analytical characteristics, such as LLOQ,  accuracy, precision, influence of matrix using dilution prior to storage and the stability of the analyte(s) in the biological matrix for the entire period of sampling, storage and processing conditions, should be  carefully  assessed  in  order  to establish the  microsampling method.     When  conventional methods have been already used in some studies and microsampling is proposed for others, a comparability of the exposure measurement, in the given matrix, between microsampling and conventional methods may be necessary. This comparison is particularly important if the conditions that the TK samples arepresented in are substantially different (e.g. dried sample from microsampling vs. liquid sample from conventional sampling). This comparability can be done in a separate pharmacokinetic (PK) study to measure area under the matrix level concentration-time curve (AUC) and/or Cmax, provided that the appropriate range of concentrations is evaluated, and

the resulting parameters compared between the two methods. This PK study, if needed, should be completed prior to conducting definitive studies that will include the microsampling procedure. A separate PK study for comparison may be omitted on a case-by-case basis and with appropriate scientific justification, for example, when using similar assay conditions to test blood, plasma or serum drawn from the same sampling site.

During   this   comparison   process,   multiple   blood   collections at a few time   points by microsampling from a few animals and their subsequent measurement of analyte concentrations can be considered in order to check the variability of sampling.   Ideally, the same matrix should be used throughout the TK studies and also in clinical studies for comparison of exposure. When different  matrices  are used in different studies, the drug concentration relationship among matrices should be defined considering appropriate factors such as haematological parameters, plasma protein binding ratio and blood/plasma (or serum) ratio of the drug, so that systemic exposure can be evaluated appropriately from each measurement using different matrices.

A3 Generally, microsampling is applicable to the majority of  pharmaceuticals   including biopharmaceuticals.  However, for all types of analytes, consideration should be given on a case- by-case basis as to whether the sensitivity of the measurement method is appropriate with the small sample volumes available.

Microsampling can be used in any type of toxicology study, such as single-dose or repeat-dose toxicology studies, and other toxicology studies (e.g. carcinogenicity, juvenile and reproductive studies).  When microsampling is applied, sampling of a representative subgroup is acceptable, as mentioned in the S3A guideline. There are published examples demonstrating no impact on key veterinary clinical pathology or pathological parameters when small volumes of blood are taken from adult animals.   However, microsampling is not warranted when the drug concentration is low and the majority or all samples are Below the Limit of Quantification (BLQ) (e.g., exposure after topical   or   inhaled   administration).     However,   when   the   bioanalytical   method   for microsampling has the same Lower Limit Of Quantitation (LLOQ) with that for conventional sample volume, microsampling can be used even if majority or all samples are BLQ.

A2 Minimizing the volume  of blood  collection  can reduce pain  and  distress  in  animals  and improve the animal welfare (refinement) of rodents and non-rodents. It can also eliminate the TK satellite group in which the TK assessment is conducted in main study animals or, reduce the number of required animals in a TK satellite group when used in rodent studies (reduction). The benefit is particularly notable for mice, since a significant number of these animals are generally used in satellite groups, in TK studies using conventional sample volumes. The main scientific advantage of microsampling is that the relationship between the safety data and drug exposure can be directly evaluated in the same animal.

A1 For the purpose of this document, microsampling is a method to collect a very small amount of blood (typically ≤50 µL) that is generally used to measure concentrations of a drug and/or its metabolites, and subsequently calculate the appropriate TK parameters. The appropriate matrices used for microsampling techniques include blood and its derived plasma or serum, which can be used in liquid or dried form for transportation, storage and subsequent analysis. Microsampling for TKs can be used in rodents and non-rodents. Microsampling in non-blood derived matrices is outside the scope of this Q&As document.