• Hemoglobin is a protein present in any bio specimen, and hemoglobin content is an important value to know for research because extreme values can create false negatives and positives for other substances in that sample.

• The IU Genetics Biobank Sub Aliquot Lab is responsible for preparing samples for research and collecting preliminary data such as the hemoglobin content. Preparing a plate for a hemoglobin assay takes two and a half hours and they must be used within twenty-four hours. These time restraints can impact the quantity of samples able to be processed as well as the ability to rectify mistakes the same day if there is an issue with the first assay.

• The Sub Aliquot Lab is interested in determining if reagents used in hemoglobin testing degrade over time and pinpoint a range in which these reagents are still viable. This is being accomplished by comparing the results from old reagents to new ones and determining the standard deviation. Positive results moving forward would allow for preparation of hemoglobin plates up to two months in advance and improve time efficiency in the lab.

• A 96 well plate with removable rows (1-12) was used to facilitate this experiment.

  • Until the conclusion and revision of the SOP, only a plate coated within 24 hours of the hemoglobin assay is permitted to be used for valid results. For this reason, the first three rows (yellow) were taken from a fresh plate to serve as the control.

• Four separately coated sets of three at various dates were used per plate during the assay.

• The procedure, run with this formatting, was completed two times a week. Plates were coated (steps 1-2 of procedure) several times a week throughout the length of the experiment to create a plate supply with a multitude of ages.

Plate Coating and Blocking

1. The first compound applied to the plate is Goat Anti-Human Affinity Purified Antibody, diluted with a coating buffer. This is incubated for one hour, and then washed out.

2. The next compound used is the blocking buffer. This is incubated for 30 minutes, and then washed out.

Standard and Sample Preparation

3. A serial dilution of the sample is applied. This is incubated for one hour, and then washed out.

4. The Goat Anti-Human HRP Conjugate, diluted with a sample diluent, is applied next. This is incubated for one hour and then washed out.

5. The TMB substrate is applied next, and incubated in the dark for 15 minutes.

6. At the end of the 15 minutes, a stop solution is applied and the hemoglobin readings are collected using a spectrophotometer [in this case, the Magellan was used]

The results of two subcategories from the data set were graphed separately to establish a consistent trend.

1. • The average standard deviation values per each coating age were compared against the average well OD values per coating age.

   • Although the slope of the line is nearly zero, indicating minimal change from the day zero values to the day forty-six values, the R² value indicates a large degree of variation.

The large degree of variation is the product of two factors: a junior scientist executing the procedure, and the volatile nature of the assay itself.

1. • Hemoglobin assays can be influenced by a great multitude of things such as room temperature, the brand of plastic used, the exact concentration of the reagents used, pipette technique, etc..

In conclusion...

• Taking into account the volatile nature of the assay, these results have been approved by the site supervisor as valid and will be considered during the modification of the current standard operating procedure for this assay.

• The R² value is of little consequence in a real world application moving forward because the purpose of the assay is to determine a hemoglobin rating of a sample based on the values measured from a standard, both of which are collected at the same time. The R value is a representation of comparison from plate to plate according to the circumstantial factors listed in prior, not the homogeneity of the results within a single plate itself.