Vitamin D testing is one of the highest-volume clinical chemistry assays performed globally. Tens of millions of 25-hydroxyvitamin D tests are run annually on automated immunoassay platforms — and every one of those assay kits requires a calibrator matrix with Vitamin D removed to background level. That matrix is Human Processed Serum (HPS) Vitamin D depleted.
This guide explains what it is, how it is produced, why the human matrix matters for assay accuracy, and what to look for when sourcing it for IVD manufacturing.
What Is Human Processed Serum (HPS) Vitamin D Depleted?
Human Processed Serum (HPS) Vitamin D depleted is pooled human serum that has undergone charcoal stripping to remove endogenous 25-hydroxyvitamin D (25-OH-D₂ and 25-OH-D₃) and 1,25-dihydroxyvitamin D to residual levels below 4 ng/mL — typically undetectable on standard immunoassay platforms.
The product is also referred to by several alternative names in the IVD industry and scientific literature:
- Vitamin D free human serum — terminology used by US suppliers including Golden West Biologicals
- Vitamin D stripped human serum — common in clinical trial and method development protocols
- Charcoal stripped serum (Vitamin D) — processing method descriptor
- 25-OH-D depleted serum — analyte-specific descriptor
- HPS Vitamin D depleted — SEQENS IVD / Avantor nomenclature, the most precise IVD industry term
Regardless of the terminology used in your specification or technical file, these products describe the same matrix — human serum processed to remove Vitamin D to below assay detection.
Why Human Serum — Not a Buffer or Synthetic Matrix?
The choice of matrix for Vitamin D calibrators is not arbitrary. Human serum is the preferred — and in many cases required — base matrix for two reasons.
Vitamin D Binding Protein (DBP) and albumin. In human serum, approximately 85% of circulating 25-OH-D is bound to Vitamin D Binding Protein (DBP), and around 15% to albumin. Only 0.03% is truly free. Most immunoassay platforms use antibodies or DBP-competitive formats that interact with total Vitamin D — bound and free. When calibrators are prepared in buffer rather than human serum, the DBP background is absent, and the assay response to spiked Vitamin D differs from the response in patient samples. This creates systematic calibration error across the clinical range.
Preparing calibrators in Vitamin D depleted human serum preserves the DBP and albumin background — ensuring the calibrator matrix matches patient sample matrix and minimising between-method bias.
IVDR matrix requirements. Under IVDR Annex I performance requirements, manufacturers must demonstrate that assay performance is established in a matrix representative of the intended sample type. For serum-based Vitamin D assays, this means calibrators must be prepared in human serum matrix — not buffer or animal serum. Vitamin D depleted human serum is the standard solution.
How Is Vitamin D Depleted Serum Produced?
The production process for HPS Vitamin D depleted involves two main steps.
Step 1 — Delipidation. Pooled human serum is treated with dextran sulphate and magnesium chloride or calcium chloride, causing lipoproteins (VLDL, LDL, HDL) to precipitate. The serum is centrifuged and filtered, removing lipids that would otherwise cause turbidity interfering with photometric measurements. Most Vitamin D is associated with lipoprotein fractions, so delipidation also partially depletes Vitamin D — but not to the low levels required for calibrator use.
Step 2 — Charcoal stripping. Dextran-coated activated charcoal is added to the delipidated serum. The charcoal adsorbs small hydrophobic molecules including Vitamin D metabolites, steroids, thyroid hormones and fatty acids. The charcoal is removed by centrifugation and filtration, leaving behind a serum depleted of the target analytes. Multiple charcoal treatment cycles may be used to achieve residual 25-OH-D below 4 ng/mL.
Step 3 — Sterile filtration and QC. The processed serum is sterile filtered at 0.2 µm and tested for residual 25-OH-D by immunoassay and/or LC-MS/MS before release. Viral screening (HIV-1/2, HBsAg, anti-HCV, Syphilis) is performed per lot.
Residual 25-OH-D Specification — What Level Do You Need?
The standard specification for HPS Vitamin D depleted is residual 25-OH-D below 4 ng/mL. This is sufficient for most immunoassay calibrator applications where the lowest calibrator in the kit is typically 10–15 ng/mL.
However, for LC-MS/MS methods — where detection limits can reach 1–2 ng/mL — a tighter residual specification may be required. Some method development protocols specify residual 25-OH-D below 2 ng/mL or below 1 ng/mL. This is achievable with additional charcoal treatment cycles but requires confirmation in the specific assay system before use.
When sourcing HPS Vitamin D depleted for LC-MS/MS applications, request lot-specific analytical data showing the residual 25-OH-D measured by the same method you will use for your assay — not just immunoassay results, which may not detect trace residual levels that interfere with mass spectrometric detection.
What to Look For When Sourcing — Key Parameters
Viral screening per lot. HPS Vitamin D depleted is a pooled human blood product. Every lot must be individually screened for HIV-1/2, HBsAg, anti-HCV and Syphilis. For IVD manufacturing, NAT screening (HIV-1 RNA, HCV RNA, HBV DNA) is increasingly required — particularly for products entering IVDR technical files. Confirm with your supplier whether NAT is standard or available on request.
Donor traceability. Complete donor traceability must be on file — not just pool-level records. This is required for IVDR Annex I documentation and for any technical file submission to a Notified Body. Some suppliers cannot provide individual donor traceability for pooled material — verify before ordering.
Lot-specific CoA with Vitamin D analytical data. The Certificate of Analysis must include the residual 25-OH-D result for that specific lot, not a generic specification. Lot-to-lot variation in residual Vitamin D is common — particularly for delipidation-only products. Insist on lot-specific measurement data.
Volume and batch consistency. For IVD manufacturing, calibrator performance is validated against a specific lot of matrix. Once validated, changing the lot requires re-validation — which is costly and time-consuming. Look for suppliers who offer batch reservation (holding a validated lot for your production lifecycle) without prepayment.
Documentation for IVDR. IVDR Annex II and Annex III technical file requirements for raw materials include: CoA, CoO, TSE/BSE statement, viral screening records, donor screening records. For ancillary materials, an ISO 13485 manufacturer certificate from the processing facility may be required. Confirm the documentation package before ordering for IVDR-regulated products.
Platform Compatibility
HPS Vitamin D depleted is compatible with all major automated immunoassay platforms used for 25-OH-D testing:
- Roche Diagnostics — Elecsys Vitamin D total assay (electrochemiluminescence)
- Abbott Diagnostics — Architect 25-OH Vitamin D (CMIA)
- DiaSorin — LIAISON 25-OH Vitamin D TOTAL (CLIA)
- Siemens Healthineers — ADVIA Centaur Vitamin D Total
- Beckman Coulter — Access Vitamin D Total
For LC-MS/MS methods, the depleted serum matrix is used for protein precipitation-based extraction — the turbidity-free, lipid-depleted base prevents ion suppression artefacts that would arise with non-delipidated serum.
How Much Do You Need?
Order quantities for HPS Vitamin D depleted vary significantly by application:
Method development / assay feasibility: 100–500 mL — sufficient for initial calibrator preparation and spike-recovery validation.
Assay validation (IVDR performance study): 1–2 L — enough for full analytical performance studies including linearity, precision, trueness and interference.
Commercial IVD kit manufacturing: 5–10+ L per production run — depending on calibrator kit format, number of calibrator levels and batch size. For high-volume Vitamin D kit manufacturers, annual requirements can reach 50–100+ L.
When ordering for manufacturing, always request more than your immediate need — reserve a full production lot to avoid mid-programme lot changes and the associated re-validation costs.
SeamlessBio — HPS Vitamin D Depleted for IVD Manufacturing
EU supplier · Passau, Germany · 100 mL to 10+ litres · Full viral screening · Lot-specific CoA · IVDR documentation · Batch reservation without prepayment · Free evaluation sample · 48-hour quote response
References
Hollis BW. Measuring 25-hydroxyvitamin D in a clinical environment: challenges and needs. Am J Clin Nutr. 2008;88(2):507S–510S.
Farrell CJL, Herrmann M. Determination of vitamin D and its metabolites. Best Pract Res Clin Endocrinol Metab. 2021;35(4):101488.
Cavalier E, et al. External quality assessment of 25-hydroxyvitamin D. Clin Chim Acta. 2014;431:60–65.
IVDR Regulation (EU) 2017/746, Annex I — General safety and performance requirements.
SeamlessBio GmbH, Passau, Germany. This article is for scientific informational purposes. For product specifications and availability, contact info@seamlessbio.de.
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