Laser-incubated immunohematological tests are faster and more sensitive than current best practice

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Researchers from BioPRIA, based at Australia’s Monash University, together with industry partner Haemokinesis, have developed the world’s first blood incubator using laser technology.

This could prevent fatal blood transfusions in critically ill patients, and can detect antibodies in pregnant women that can kill a foetus.

According to results published in Nature’s Scientific Reports, these findings could bring pre-transfusion testing out of the pathology lab to point-of-care, with blood incubation time slashed to just 40 seconds, compared with the industry gold standard of five minutes.

This breakthrough has the potential improve the pre-transfusion testing of millions of patients undergoing blood transfusions across the world, especially those having major surgery, going into labour, or causalities of mass trauma and individual trauma.

The detection of immunoglobulin G (IgG) antibodies requires incubation at 37°C, often for up to 15 minutes.

But current incubation technology relies on slow thermal procedures, such as heating blocks and hot-water baths.

This delay adds to pathology costs and turnaround time, which substantially affects a patient’s chance of survival.

To address this problem, BioPRIA’s blood diagnostics team developed a laser incubation model where a targeted illumination of a blood-antibody sample in a diagnostic gel card is converted into heat, via photothermal absorption.

The laser-incubator heats the 75 μL blood-antibody sample to 37°C in under 30 seconds. Most importantly, no significant damage is detected to the cells or antibodies for laser incubations of up to 15 minutes.

The study was led by Dr. Clare Manderson from the Bioresource Processing Institute of Australia (BioPRIA), located within the Department of Chemical Engineering at Monash University, in conjunction with blood diagnostics manufacturer Haemokinesis.

“Laser incubation can be extremely valuable when time and accuracy is vital, especially in critical and emergency settings—like mass trauma—where pre-transfusion testing needs to be performed quickly in order to save lives,” Dr. Manderson said.

“We show that red blood cells act as photothermal agents under near-infrared laser incubation, triggering rapid antigen-antibody binding with no significant damage to the cells or antibodies for up to 15 minutes.

“This study demonstrates laser-incubated immunohaematological testing to be both faster and more sensitive than current best practice, with clearly positive results seen from incubations of just 40 seconds.”

For this study, researchers explored the roles of incubation time and temperature of the IgG anti-D antibody and the Rh blood group system’s D antigen, which indicates the positive or negative attributed to a person’s ABO blood type group.

Experimental laser chamber and gel card indirect antiglobulin test (IAT). (a) Our laser incubation chamber illuminates a blood-typing gel card column containing a red blood cell (RBC)-antibody suspension with infrared laser light (980 nm). (b) Experiment steps: (1) RBC and antibody solutions are added to the gel card upper chamber. (2) RBC-antibody suspension is incubated by laser photons entering from above. (3) Gel card is centrifuged to mix the antibody-bound or unbound RBCs with the anti-antibody (IgG) and pass through the gel column. (4) Agglutinates and RBC positions are observed. (c) Scoring the result: positions of the agglutinates indicate strength of result.
Experimental laser chamber and gel card indirect antiglobulin test (IAT). (a) Our laser incubation chamber illuminates a blood-typing gel card column containing a red blood cell (RBC)-antibody suspension with infrared laser light (980 nm). (b) Experiment steps: (1) RBC and antibody solutions are added to the gel card upper chamber. (2) RBC-antibody suspension is incubated by laser photons entering from above. (3) Gel card is centrifuged to mix the antibody-bound or unbound RBCs with the anti-antibody (IgG) and pass through the gel column. (4) Agglutinates and RBC positions are observed. (c) Scoring the result: positions of the agglutinates indicate strength of result.


Anti-D is the most common antibody, and is present in a person’s plasma. It’s the biggest cause of haemolytic disease of the foetus and newborn—a blood disorder that occurs when the blood types of a mother and baby are incompatible.

Accurate testing for pregnant women’s antibodies is vital to save the life of the foetus.

Blood group type is based on the presence of antigens on the surface of the red blood cell membranes, consisting of proteins, glycoproteins, glycophorins, glycolipids and polysaccharide macromolecules forming roughly 346 known blood groups.

“Giving blood transfusions to people isn’t as simple as giving O-negative to anybody.

The ‘universal donor’ of O-negative blood can seriously harm a lot of people, even kill them.

The world of pre-transfusion of blood group typing is huge, and it’s really important that it’s done quickly and accurately to help save lives,” Dr. Manderson said.

“For the patient, it can mean that if there’s a critical blood-loss scenario and they’re in desperate need of a transfusion, they need to have their blood group typed and antibody screened as quickly as possible. We’re aiming to bring that down to seconds instead of tens of minutes.”

Blood transfusion is a critical treatment for a variety of haematological conditions, including cancer chemotherapy, bleeding trauma, childbirth and major surgery. Transfusion reactions are common if the recipient and donor aren’t correctly matched.

More than 1.2 million blood components are transfused each year in Australia, and 21 million in the US.

While the technology isn’t yet commercially available, Haemokinesis holds a patent for this innovation.


Blood transfusion is a critical treatment for a variety of haematological conditions, including cancer chemotherapy (1.7 million new patients/year in US), sickle cell disease treatments (100,000 patients/year in US), bleeding trauma, including childbirth (4 million births/year in US), and major surgery.

Over 21 million blood components are transfused in the US alone every year; each having the potential for fatal haemolytic transfusion reactions if recipient and donor are not accurately matched.

Furthermore, pregnant women often have a different blood group to that of their unborn child whereby blood-borne antibodies capable of crossing the placenta can cause haemolytic disease of the foetus and newborn (HDFN).

Immunohaematological tests, where blood group is typed (determined) and antibodies are screened for and identied, must be performed for all patients, donors and preg-nant mothers to prevent possible fatalities.

Blood group type is based on the presence of antigens on the surface of the red blood cell (RBC) membranes, consisting of proteins, glycoproteins, glycophorins, glycolipids and poly-saccharide macromolecules, forming over 346 known blood groups1.

For each, specific antibodies can be present in a person’s plasma.

This presents an enormous immunohaematological industry with hundreds of millions of tests performed globally each year.Identication of antigens and antibodies often requires incubation at human body temperature of 37 °C. Depending on the method, this incubation step can take 5 to 30 minutes.

This delay adds to pathology cost and turnaround time and can endanger a patient’s life. Current incubation methods consist of dry-air incu-bators (ovens), heating blocks or hot water baths which all rely on the relatively slow thermal energy transfer methods of conduction and convection.

Electromagnetic radiation-based blood warming technologies using radio- and microwaves have been used since the 1960s in pre-transfusion blood warming for the prevention of patient hypothermia.

However, these techniques have lacked the very precise temperature control or fast and uniform heating rates required for sensitive immunohaematological reactions.

Furthermore, for use with the highly sensitive gel card diagnostics, microwave incubation which lacks targeted and localised heating, would heat the whole gel card, potentially damaging the gel matrix, making it an unsuitable method for immu-nohaematological tests.

Optical heating via laser-light incubation provides an opportunity to not only rapidly heat directly inside the sample volume, but also to preferentially heat the surface of the red blood cell (erythrocyte) and activate antigen-antibody binding.

Here, we present laser-based incubation for the photothermal heating of red blood cell and antibody samples in traditional gel cards, where the optical absorption properties of blood and water produce thermal energy (heat) via non-radiative decay processes.

By selectively controlling the power, wavelength and positioning of the laser light, the incubation time can be considerably reduced without signicant damage to the cells or biomolecules.Light-to-heat converters have been explored in biomedicine for a range of applications, including modern therapies, imaging and biosensing.

Infrared lasers have also been used for heating of 50 µL droplets in polymer-ase chain reaction (PCR) studies7,8 where in our study, the RBCs may act as photothermal agents.Immunohaematological tests use the specic binding (complexing) of antibody to antigen (epitope) to form RBC agglutinates (cell aggregates) to indicate a positive result. Immunoglobulin M (IgM) antibodies are pentamers and are able to bridge the RBC’s native repulsion charge to form agglutinates directly; and are used for the detec-tion of most antigens.

However, the monomer immunoglobulin G (IgG) antibodies, whose presence must be detected in pregnant mother or patient plasma, cannot directly agglutinate cells.

They require secondary anti-IgG antibodies to bridge the IgG sensitized cells — forming the indirect antiglobulin test (IAT) or Coombs test.

Sensitization of ‘warm reacting’ IgG antibodies occurs best at 37 °C, human body temperature, requiring a minimum 5 to 15 minute incubation, with longer incubation periods often enhancing the reaction and reducing the so-called ‘cold reacting’ IgM antibodies from returning false positives.

In this proof of concept study, we explore the roles of incubation time and temperature of the Rh blood group system’s D antigen and the IgG anti-D antibody. It is the presence of the RhD epitope, a multipass transmembrane protein, which indicates the positive or negative attributed to a person’s ABO blood group type.

Anti-D is most commonly present as IgG in a person’s plasma. It is the most important type of antibody to test for in transfusion medicine, and is the biggest cause of HDFN.

Complexation of the epitope (antigen) and paratope (antibody binding site) has a temperature dependent binding rate however the literature is lacking compelling evidence and the issue has not been investigated in decades.

This provided an opportunity to enhance the current standards of immunohaematological testing with contemporary science.

Here, we investigate the potential of laser incubation as a rapid and sensitive method for immunohaematol-ogy.

We optimised a laser incubation system and determined the parameters required for effectively and stable heating the RBC-antibody solution.

The role of incubation time and temperature are demonstrated to play key roles in strength of result.

Furthermore, laser incubation is shown to both enhance agglutination strength for weak antibody solutions and reduce required incubation time for successful positive results. We show that the laser preferentially heats the RBC surface’s micro-environment, inducing RBC-antibody complexation, via the mechanisms of infrared light absorption and scattering by RBC solutions.

Finally, we demonstrate that the laser does not induce RBC damage for incubations of 15 minutes or less and that antibody potency is preserved for up to 30 minutes in this optimised system.


More information: Clare A. Manderson et al, Photothermal incubation of red blood cells by laser for rapid pre-transfusion blood group typing, Scientific Reports (2019). DOI: 10.1038/s41598-019-47646-y

Journal information: Nature , Scientific Reports
Provided by Monash University

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