+ Precautions to prevent transmission of infectious agents when handling blood samples

All staff members have to be trained about the risks of exposure to blood borne pathogens prior to handling blood samples. Blood borne pathogens are pathogenic microorganisms that are present in human blood and can cause disease in humans. Proper procedures for handling, transportation, storage, and disposal of blood samples have to be followed. Page 66 of this Guideline from the Healthcare Infection Control Practices Advisory Committee (HICPAC) describes Standard Precautions.

+ Prevention of zinc contamination of samples

Zinc is present in serum and plasma only in very low concentrations. Thus, any contamination from external sources has to be avoided as they can dramatically change the results. The procedures described below should be followed to reduce the risk of contamination.

+ Practices and supplies to avoid contamination

• Regularly clean the workspace (phlebotomists’ work area, laboratory work space, hood, supplies, etc). Keep it free of dust.
• Throughout blood collection and processing, only powder-free gloves should be used as the powder can be a source of zinc contamination.
• Only supplies free of zinc can be used for blood collection and sample processing. This can be achieved by purchasing certified trace-metal free supplies and/or by analyzing the zinc content of laboratory supplies prior to their use. List of trace element-free supplies.

+ Blood collection technique

• Clean subject’s skin with alcohol at site of antecubital vein or the dorsal area (back of the hand)
• Restrict occlusion of subject’s arm with tourniquet < 1 minute
• Draw blood using stainless steel needle, and collect into trace element-free evacuated blood collection tubes (trace element-free supply list).
• A strong light is helpful in allowing the phlebotomist to visualize the vein. In settings without electricity, a battery-operated head lamp worn by the phlebotomist can be very useful to increase visibility.

The blood should be collected following a strict protocol that controls the time of day and fasting status of the participant. If fasting is not possible, then where feasible, each subject could be provided with a standard snack (e.g., biscuit or breast feeding) and the blood drawn after a standardized time interval (e.g., one hour).

To be able to control for these factors in the analyses, the following information should be recorded:

• Time of previous meal (including breast milk, for infants)
• Time of blood draw
• Time of centrifugation and separation of serum or plasma

When drawing blood from young children, an option is to use a local anesthetic cream to reduce the pain of the blood draw. Any such product should be tested for zinc content prior to use. One product that is a vaso-dilator and found to be free of zinc is methocaine (Ametop[TM]).

+ Processing samples

Once the blood is obtained, a cold chain (2 to 10 °C) must be maintained. Samples should be stored and/or transported in a refrigerator or a portable cool box (electric or with ice packs). It is recommended to keep a temperature log for temperature documentation throughout the process.

• When the storage temperature is 2 to 10 °C, the sample is stable for up to 24 hours.
• If the cold chain cannot be guaranteed until the sample is processed, it is important to separate the serum or plasma from the red blood cells within 20-30 minutes.
• These precautions are important to prevent zinc being transferred from blood cells to serum or plasma, which leads to an artificial increase in the zinc content.
• After centrifugation, the plasma or serum should be transferred to a zinc-free polypropylene tube and stored in the refrigerator or freezer until analyses.

+ Aliquoting of samples in the field laboratory

To avoid external zinc contamination, we recommend that the samples should be aliquoted under a hood to protect from dust and other sources of contamination. If no laboratory hood is available, simple hoods can be built in country at low-cost. Pipettes and pipette tips used to aliquot samples should be protected from dust by storing in a zip-lock sealed plastic bag. Use of pipette tips that have not been stored in a sealed plastic bag should be avoided. Pipettes should be kept clean and, when necessary, they should be cleaned according to the manufacturer’s instructions.

Centrifuge blood sample at 2000-3000 × g for 10 minutes to separate serum or plasma. If centrifuging with a portable centrifuge in the field, you need to make sure the red blood cells are being completely separated from the plasma. Discard any severely hemolyzed samples and make a note of those samples with some evidence of hemolysis.

Store plasma or serum samples in polypropylene tubes. Avoid sample storage tubes with silicone washer seals; the seals may be a source of contamination. For long-term storage in a -20 °C or -80 °C freezer, store samples in a sealed plastic bag containing ice cubes to avoid evaporation.

Example of hoods built by carpenters
Example of hoods built out of plastic boxes

+ Sample analyses

Dilute sample for zinc 5 to 10-fold in solvents such as 0.05N aqueous hyperpure HCl. Read sample zinc concentration using an available instrument with appropriate standard dilutions, in-house quality controls, and Standard Reference Materials.

Example of a laboratory protocol for flame atomic absorption spectrophotometry (AAS)

Example of a laboratory protocol for inductively coupled plasma optical emission spectrometry (ICP-OES)

+ Reference laboratory

Inter-laboratory comparison with a reference laboratory is highly recommended. The list below is not meant to be exclusive and is not an endorsement of these laboratories.

United Kingdom National External Quality Assessment Service (NEQAS)
www.ukneqas.org.uk

BIO-RAD External Quality Assurance Services (EQAS)
www.qcnet.com

+ Statistical analyses

Prior to analysis, the distribution of plasma or serum zinc results should be reviewed to check for outliers (implausibly low or high values). One approach is to set statistically defined-cutoffs for outliers: for example, >3 standard deviation (SD) from the mean of age-specific US NHANES plasma zinc distribution (Pilch and Senti, 1984).

Because plasma zinc concentration is reduced in the presence of inflammation, it is recommended to include the analyses of acute-phase proteins. C-reactive protein (CRP) (biomarker of acute inflammation) and a-1 acid glycoprotein (AGP) (biomarker of chronic inflammation) can be used for that purpose. When elevated acute-phase proteins are found, the corresponding zinc concentration can be adjusted statistically. If the time of day and time since previous meal cannot be standardized during data collection, these variables should also be controlled for during the data analysis. Please consult with a statistician for further details.

Use appropriate cutoffs depending on characteristics of study population (see table 1 below) (Brown et al, 2004).

Table 1: Suggested lower cutoffs for serum zinc concentration (ug/dL) by age group, sex, time of day and time since last meal

1For conversion to µmol/L, divide by 6.54.
2 Fasting is defined as no food or beverage consumption for at least 8 hours.