Systems, Devices and Methods for Sequential Analysis of Complex Matrix Samples for High Confidence Bacterial Detection and Drug Susceptibility Prediction Using a Flow Cytometer

A method of using a flow cytometer for testing a sample of a body fluid for the presence of bacteria, and optionally determining sample response to at least one antibiotic, comprising:

• adjusting dilution of the sample to a predetermined concentration;
• dividing the diluted sample into at least two batches including a time 0 baseline (T0 baseline) batch and a time 1 control (T1 control) batch;
• testing the T0 baseline batch at time T0 with the flow cytometer to obtain T0 enumerative baseline bacterial values relating to measured characteristics of the T0 batch;
• culturing the T1 control batch in growth media from time 0 to time 1;
• testing the T1 control batch at time 1 with the flow cytometer to obtain T1 enumerative control bacterial values relating to measured characteristics of the T1 sample;
• and
• comparing the T1 control values to the T0 baseline values to determine a growth ratio of samples containing bacteria.

A method of compensating for inaccuracies in flow cytometer enumeration of particles of interest in fluid samples, comprising:

• including a known concentration of a test-enumerative compensator (TEC) particles in the sample to be enumerated, said TEC particles having known flow cytometric scatter and fluorescence characteristics;
• enumerating the TEC particles with the sample enumeration by the flow cytometer;
• determining a compensator factor based on the enumerated TEC particle value as compared to the known TEC particle concentration in the sample tested;
• and
• adjusting the sample test enumeration value by said compensator factor.

A system for automated testing a sample of a body fluid for the presence of bacteria, and optionally determining sample response to at least one antibiotic, the system comprising:

• fluid handling device including an automated pipetting system for distributing fluid samples among wells of a well plate;
• incubator configured to culture samples in well plates received from the fluid handling device;
• plate transport device configured to deliver well plates containing samples to the incubator from the fluid handling device and return well plates from the incubator to the fluid handling device;
• flow cytometer configured to enumerate cell counts in samples provided by the fluid handling system;
• processor and memory, the processor configured execute instructions stored in the memory to control the system in accordance with said instructions, wherein the stored instructions cause the system to —
• distribute a portion of the sample to at least a first test well;
• enumerate the sample portion from the at least first test well to determine a total bacteria count;
• adjust dilution of the sample with growth media to a predetermined concentration based on the total bacteria count;
• divide the dilution-adjusted sample into at least wells including a time 0 baseline (T0 baseline) well and a time 1 control (T1 control) well;
• enumerate the sample in the T0 baseline well at time T0 to obtain T0 enumerative baseline bacterial values relating to measured characteristics of the sample in the T0 well;
• deliver the sample to the incubator;
• culture the sample in the T1 control well from time 0 to time 1;
• return the sample to the fluid handling device after culturing;
• enumerate the T1 control batch at time 1 with the flow cytometer to obtain T1 enumerative control bacterial values relating to measured characteristics of the T1 sample;
• and
• compare the T1 control values to the T0 baseline values to determine a growth ratio of samples containing bacteria;
• and
• a graphical user interface communicating with at least the processor allowing user interaction with the system.

The system according to claim 25, wherein said instructions stored in memory further cause the system, at or approximate to time 0:

• distribute dilution-adjusted portions of the sample to at least to at least one AT well inoculated with an antibiotic of interest;
• enumerate the sample portion from the first well with a flow cytometer to determine AT enumerative bacterial values relating to measured characteristics of the sample in the at least one AT well;
• and
• compare the AT enumerative values to the T1 control values to determine a response of the sample to the antibiotics of interest.