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Pre-analytical variables influence zinc measurement in blood samples.

Author(s): Killilea DW, Schultz K

Publication: PLoS One, 2023, Vol. 18, Page e0286073

PubMed ID: 37713369 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared zinc levels of zinc between capillary and venous blood, plasma and serum, and specimens collected in tubes from Becton Dickinson (BD) and Sarstedt. Zinc levels were also compared in plasma/serum from blood stored at 4°C, 20°C or 37°C for 1 h followed by storage at 4°C for a total of 4 or 24 h.  Zinc Levels were also compared between specimens from males and female volunteers. Non-fasting blood was collected between 10 am and noon from sixty healthy volunteers first by fingerstick (capillary blood) and then by venipuncture.  The volunteers’ hands were pre-warmed in a water bath for 10-20 min before collection of capillary blood from standing volunteers. Capillary blood was collected by applying a high flow lancet to the thumb of the non-dominant hand and, when necessary, the third or fourth finger. The first drop was discarded and then pooling drops were collected into 1-4 BD Microtainer K₂EDTA Plasma Blood Collection Tubes (BCT) followed by 1-4 Sarstedt Microvette K₃EDTA Plasma BCT as volume allowed.  Once all capillary blood tubes were collected, specimens were inverted 4-6 times and placed on wet ice. Venous blood was collected from the antecubital vein of the non-dominant arm using a Sarstedt Safety 21-guage Multifly Needle with a Multi-Adaptor into a Sarstedt Monovette serum BCT containing clot activator, a Sarstedt Monovette K₂EDTA plasma BCT, eight BD Vacutainer serum BCTs with clot activator, and finally eight BD Vacutainer K₂EDTA plasma BCTs; a few drops of blood were discarded between tube types.  After blood collection, tubes were inverted 8-10 times and placed on wet ice and all specimens were transported to the lab (an average duration of 10-20 min following collection, all <60 min). To investigate the effects of storage, matched BD Vacutainer plasma and serum tubes were placed at 4°C, 20°C, and 37°C for 1 h, after which, all tubes were placed at 20°C and 37°C. After 4 and 24 h, one tube of each kind for each initial storage temperature? was analyzed. Plasma was separated from EDTA blood by centrifugation at 800 g for 15 minutes at 5°C immediately or after storage. Serum was separated by centrifugation at 800 g for 15 minutes at 5°C. Plasma and serum were centrifuged again at 4000 g for 5 min at 5°C and the supernatant transferred to polypropylene tubes and stored at -70°C until analysis. Specimens were thawed at room temperature and the degree of hemolysis was quantified using Drabkin’s assay. Zinc levels were quantified by inductively coupled plasma optical emission spectrometry.

   Summary of Findings:

   Two of the specimens (one in a BD plasma vacutainer and one plasma microtainer, both processed without delay) were found to have >100 mg/dL hemoglobin and thus were excluded from analysis.  Zinc levels were comparable in plasma from male and female volunteers (0.664 ± 0.095 mg/L and 0.633 ± 0.070 mg/L, respectively). Zinc levels were 8% higher in BD capillary than BD venous plasma (capillary: 0.700 ± 0.094 mg/L versus 0.646 ± 0.084 mg/L, respectively; p<0.0001). Zinc levels were 5% higher in BD serum than BD plasma (0.679 ± 0.092 mg/L versus 0.649 ± 0.083 mg/L, respectively; p<0.0001). Zinc levels were comparable between venous plasma from BD Vacutainers and Sarstedt Monovettes venous plasma (0.647 ± 0.083 mg/L and 0.644 ± 0.083 mg/L, respectively) and for serum from BD Vacutainers and Sarstedt Monovettes (0.679 ± 0.092 mg/L and 0.670 ± 0.089 mg/L, respectively); however, zinc levels were 6% higher in capillary plasma from BD microtainers than Microvettes (0.688 ± 0.072 mg/L versus 0.652 ± 0.084 mg/L, respectively; p<0.0001). An ANOVA identified a significant effect of blood storage temperature (during the first hour of storage) and duration on zinc levels in plasma (p=0.0007 and p<0.0001, respectively) and serum (p = 0.0061 and p<0.0001, respectively).  Plasma and serum from blood stored at 20°C or 37°C for one hour before storage at 4°C for 3 h (or 21 h) had significantly higher zinc levels than plasma/serum from blood that was processed immediately (p<0.05, all). While zinc levels were higher in plasma and serum from blood that was stored at 4°C for ≥4 h compared to plasma/serum that was isolated immediately, the difference was only significant when blood was stored for 24 h (p<0.05, both). Zinc levels were classified as low (<0.65 mg/L) in 32 of the 60 specimens collected in BD plasma vacutainer tubes. Importantly, the number of volunteers whose zinc level was classified as low varied depending on whether the specimen was venous or capillary, plasma or serum, and the tube manufacturer.  

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Electrolyte/Metal	Inductively coupled plasma mass spectrometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Biospecimen Acquisition	Anatomical location of blood draw	Anticubital vein Fingertip
   Biospecimen Acquisition	Type of collection container/solution	BD Microtainer K2EDTA Plasma BCT Sarstedt Microvette K3EDTA Plasma BCT Sarstedt Monovette Serum BCT with Clot Activator Sarstedt Monovette K2EDTA Plasma BCT BD Vacutainer Serum BCTs with Clot Activator BD Vacutainer K2EDTA Plasma BCTs
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Storage temperature	4°C 20°C for 1 h followed by remaining 3 or 23 h storage at 4°C 37°C for 1 h followed by remaining 3 or 23 h storage at 4°C
   Storage	Storage duration	0 h 4 h 24 h
   Biospecimen Acquisition	Method of fluid acquisition	Finger/heel prick with warming Venipuncture

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