Vertical flow assay device and method for determination of hemoglobin concentration

A method of fabricating an assay device for determining a concentration of hemoglobin in a blood fluid sample, the method comprising:

• coating a solution containing a cell lysing reagent onto to a separation membrane, wherein the cell lysing reagent is present on the separation membrane in an amount greater than 200 micrograms/square centimeter to less than 675 micrograms/square centimeter based on the dry weight of the cell lysing reagent on the separation membrane after the solution has dried on the separation membrane;
• allowing the solution to dry on the separation membrane;
• and
• positioning a detection membrane downstream from the separation membrane, wherein the detection membrane is configured to elicit a quantifiable response in the presence of hemoglobin in the blood fluid sample, wherein the quantifiable response corresponds to an amount of hemoglobin present in the blood fluid sample, wherein the detection membrane is asymmetric and has a first plurality of pores located towards an upstream side of the detection membrane and a second plurality of pores located towards a downstream side of the detection membrane, wherein the first plurality of pores have an average pore size that is larger than an average pore size of the second plurality of pores.