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In Vitro Hemolysis & How to Prevent it

What Is Hemolysis?

Hemolysis is the disruption or destruction of red blood cells (RBCs). Whether via natural necrosis at the end of the RBC life cycle or via abnormal breakdown of cellular membranes due to disease or experimental procedure, when hemolysis occurs, the damaged RBCs release hemoglobin and spill their cellular contents into their environment. Hemolysis often presents in patients with symptoms ranging from shortness of breath to chest pain and fatigue; establishing a diagnosis of hemolysis in a patient is contingent on performing in vitro hemolysis studies in hospital labs.

In vivo hemolysis occurring within the body is a serious pathological concern. Red blood cell lysis can be a cause of disease or a symptom of an underlying biological disorder in a patient. Lysing of RBCs can arise from several anemias due to infection, immunodeficiencies, or liver failure. Moreover, hemolysis itself can lead to the buildup of excess hemoglobin in the blood, which can cause jaundice, clotting, heart attacks, and strokes.

In kind, hemolysis in patient blood samples poses critical risks to effective medical diagnosis and experimental analysis, with any number of lab tests adversely affected by hemolysis. Degrading the viability of samples and skewing the results of analyte detection, hemolysis is the leading cause of sample rejection in testing and research—as many as 20% of Emergency Department samples are contaminated due to hemolysis, and hospital labs often must re-run bloodwork with in vitro hemolysis assay protocols that impede timely and effective treatment for patients.

What Is In Vitro Hemolysis?

While in vivo hemolysis is related to patient pathology, in vitro hemolysis often occurs due to testing procedures or during handling and processing of blood samples. As such, minimizing the causes of hemolysis in blood samples through optimizing research protocols is essential to increasing experimental accuracy, therapeutic efficacy, and, ultimately, patient safety.

Common causes of in vitro hemolysis include improper specimen collection techniques (site-selection/preparation/methodology of venipuncture in drawing blood samples), as well as mixing, transportation, and processing of blood samples in the lab. Teaching clinical staff careful best practices for sample collection and handling can reduce the likelihood of hemolysis when drawing blood from patients or processing blood samples.

Clinical research methodology also influences the occurrence of in vitro hemolysis. Experimental factors leading to hemolyzed blood samples involve the agar medium used to cultivate cell cultures, the type and concentration of blood sample used in the lab, the period of incubation for the cell cultures, the presence of air or oxygen in the culture environment, and even the placement of cell colonies on culture plates. As such, improving procedures for blood sample analysis can reduce the likelihood of in vitro hemolysis occurring during testing and research.

How Are Lab Tests Affected by Hemolysis?

If a blood sample is only slightly hemolyzed, proper hemolysis assays and removal of excess hemoglobin or cellular debris might be able to salvage the sample. That said, grossly hemolyzed cell populations adversely affect measurement and analysis in laboratory testing. Such moderate or severe hemolysis can inflate or decrease the levels of analytes being measured in a specimen as well as change the pH of reactions occurring within a blood sample during therapeutics testing—these false readings can lead to costly errors in analysis and delays in treatment.

For example, hemolysis can cause biases in Cardiac troponin [cTn] levels, interfering with the detection of markers of several heart conditions in patients. Likewise, hemolyzed blood samples exhibiting false levels of various analytes including Aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatinine, alkaline phosphatase (ALP), or potassium present significant impediments to accurate diagnosis and timely treatment in hospital patients.

Because of the prevalence of contamination from RBC lysis, labs must implement in vitro hemolysis studies as part of their sample analysis methodologies to identify lysing and mitigate its consequences. While hemolysis can be casually inferred from a pink-or-reddish hue in blood or plasma samples, more rigorous hemolysis assays are necessary to determine the severity of hemolysis (and therefore the viability of a sample) with confidence.

Preventing In Vitro Hemolysis

As the issue of detecting and preventing hemolysis is crucial to diagnosis and testing, optimizing methodologies to reduce the number of blood samples that are hemolyzed in vitro is an urgent concern. While preventing in vivo hemolysis is a pathological issue beyond the scope of this article, there are several methods to improve in vitro hemolysis assay protocols and mitigate the likelihood that experimental procedures will cause blood samples to hemolyze.

As far as specimen collection goes, choosing and properly preparing a viable site for venipuncture in a patient is the first step to reducing the risk of clinical hemolysis. Selecting an easily accessible antecubital vein and allowing the site to dry properly before drawing blood are two ways to diminish hemolysis rates during sample collection. Limiting the length of time that a tourniquet is applied to a patient’s arm while drawing blood to less than one minute can also help to minimize trauma to the site that could cause hemolysis. As well, the choice of the needle, handling of the syringe plunger, probing for a suitable vein, and catheter tubing connections all factor into reducing the risk of hemolysis during sample collection.

Once a blood sample has been collected, spectrophotometric measurement of hemoglobin levels in a blood sample population (referred to as the H-index) is an industry gold standard for consistent, accurate hemolysis recognition. However, typical protocols are unable to assess the H-index of whole-blood specimens, so identifying hemolysis and determining whether a sample was hemolyzed in vivo in the patient or in vitro during sample preparation remain extremely complex tasks.

Hemolysis induced from mechanical trauma during handling and transportation of blood samples is one common cause of in vitro hemolysis in the lab. Reducing the length, speed, complexity, and a number of transports per specimen can help diminish the risk of hemolysis. Likewise, avoiding excessively hot or cold transport environments, as well as temperature fluctuations, can help prevent RBC lysis in collected samples.

To conduct analysis of blood specimens or test the effects of therapeutics on cell populations, samples often need to be mixed with additives and undergo mixing processes to distribute compounds throughout the sample. In kind, RBCs must often be separated and removed from a heterogeneous blood sample in order to study them in isolation. Both mixing and isolation processes can cause physical trauma to the cells in a sample that can cause in vitro hemolysis to occur.

Red Blood Cell Depletion With Akadeum’s BACS Microbubbles

Akadeum Life Sciences has developed an affordable and easy-to-use Buoyancy Activated Cell Separation [BACS] technology for gently separating target cells from a sample population. The BACS mechanism involves using functionalized microbubbles that bind to target cells, lifting the bound targets to the surface of the sample container where they can be collected or removed.

Akadeum’s BACS products can be used to remove unwanted cells (like RBCs) and cellular debris from a sample quickly and easily, leaving behind a highly viable cell population for downstream analysis. Akadeum’s Human Red Blood Cell Depletion Microbubbles remove up to 99% of RBC contamination from PBMC samples in a fast and easy workflow that maintains the health and physiology of delicate cells of interest.

Learn more about RBC Depletion in PBMCs and Dissociated Tissue Samples

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