Table 2. DLD microchip processing of immunostained whole blood resulted in high recovery of WBCs including major WBC subsets, with >99% depletion of RBCs.

Experiment	Fraction Analyzed	Volume (μL)a	RBC Count (× 106 Cells/μL)	%RBC Depletedb	WBC Count (× 103 Cells/μL)c	Total % WBC recoveryd	% Granulocytesg	% Monocytesh	% Lymphocytesi	% B-Cellsj	% T-Cellsk	Run Time (Mins)
1	Input	200	1.99	–	2.9	97+/−5%	59.9	5.4	34.7	15.0	74.4	14.3
	Product	181	0.01	99.50%	3.1		61.0	5.5	33.5	13.6	76.3
2	Input	200	1.75	–	3.6	105+/−4%	68.0	4.9	27.1	18.5	75.3	12.7
	Product	193	0.01	99.43%	3.9		67.5	5.0	27.6	17.9	76.3
3	Input	200	1.92	–	2.5	98+/−7%	67.9	6.7	25.4	19.5	71.9	22.0
	Product	258	0.02	98.96%	1.9		68.7	7.0	24.2	17.7	71.7
4	Input	200	1.85	–	2.7	101+/−5%	71.2	4.9	23.9	26.4	58.4	14.3
	Product	176	0.01	99.46%	3.1		71.3	4.0	24.8	28.2	57.2
5	Input	200	2.13	–	2.4	103+/−6%	70.7	6.2	23.1	10.4	83.2	18.5
	Product	215	0.01	99.53%	2.3		72.0	5.6	22.4	9.1	84.2
6	Input	200	2.09	–	2.3	107+/−6%	71.2	5.3	23.6	10.2	83.8	11.7
	Product	223	0.02	99.04%	2.2		71.9	5.9	22.2	9.5	84.0
Mean Depletion or Recovery or Run Timee				99.32+/−0.10%		102+/−2%						15.6+/−1.6
Mean Difference (Product vs. input)f							0.6+/−0.3%	-0.1+/−0.2%	−0.5+/−0.4%	−0.7+/−0.5%	0.4+/−0.4%

^alnput and Output volumes as in Table 1.

^bDifference of Coulter Counter RBC count in Input vs Product (column 4) divided by Coulter Counter RBC count in Input. For the Products, the RBC counts were at the lower detection limit of the Coulter Counter and hence are imprecise.

^c,dAs in Table 1.

^eMean +/−standard error of the mean (SEM).

^fProduct value minus Input value (mean +/−SEM).

^gGranulocytes were identified by flow cytometry as CD45^low7CD3⁻/CD19⁻ signals with characteristic high FSC and SSC.

^hMonocytes were identified as CD45^high/CD3⁻/CD19⁻ with characteristic intermediate FSC and SSC.

ⁱLymphocytes were identified as CD45^high/CD3^{+ or −}/CD19^{+ or −} with characteristic low FSC and SSC.

^jB cells were identified as CD45^high/CD3⁻/CD19⁺ with FSC and SSC of lymphocytes.

^kT cells were identified as CD45^high/CD3⁺/CD19⁻ with FSC and SSC of lymphocytes.

^g,h,i,j,kPercent cells of each subtype was calculated using the number of events for that cell population divided by the total number of viable cell events, as determined by flow cytometric FSC/SSC-gating.

Blood from five different donors was analyzed in six replicate experiments. All experiments performed over four weeks, using a fresh microchip for each experiment. The microchips were emptied by following the blood sample with an air plug as a “flush” to clear the microchip and tubing of cells. Prior to flow cytometric analysis, all Input and Output samples were subjected to RBC lysis, to provide comparably treated cells for flow cytometry. For RBC lysis, 100 μL filtered blood was combined with 900 μL 1 × FACS lysing solution (Becton Dickinson, San Jose, CA), vortexed and incubated (RT, 15 min). Following centrifugation (1,000 × g, 10 min), the supernatant was removed and the WBC-enriched pellet resuspended in 1 mL run buffer. After an additional centrifugation, the supernatant was removed and the WBC-enriched pellet resuspended in 250 μL run buffer. This procedure required ∼45 min.