A Precision Medicine Tool for Measurement and Monitoring of Hemoglobin S in Sickle Cell Disease Patients Receiving Transfusion Therapy

Summary

We have developed a point-of-care immunoassay to rapidly quantify hemoglobin S (HbS) levels during transfusion therapy for sickle cell disease (SCD) patients. By applying a small amount of treated blood to the device, the healthcare provider can determine the %HbS in a SCD patient to immediately inform clinical decisions.

Abstract

Sickle cell disease (SCD) causes many severe health complications, including anemia, stroke, and acute chest syndrome. Red blood cell transfusion is the most commonly used therapy to treat or prevent these devastating complications. Other therapies include hydroxyurea therapy as well as bone marrow transplantation. Chronic intermittent transfusion is especially indicated to prevent recurrent strokes. However, transfusion therapy is associated with significant adverse effects (e.g., alloimmunization and iron overload). The point-of-care (POC) lateral flow immunoassay used here quantifies the %HbS in 15 min using a small patient blood sample. Utilizing this information, the healthcare provider is able to adjust blood transfusion volume for SCD patients to achieve the desired target HbS (most often <30%), while reducing the risk of transfusion-related complications. When compared to laboratory hemoglobin electrophoresis data for 38 whole blood samples, the POC test performed with very high correlation and linear fit (slope = 0.9949, R² = 0.9751). The strong agreement between the two hemoglobin S percentage (%HbS) quantifying methods shows that 89.5% of samples fall within ±5.2% HbS bias. The calibration for quantifying %HbS is built into the device to allow for an automated quantification of %HbS. This time- and cost-effective POC test thus allows the healthcare provider to make timely informed decisions when treating SCD patients, using accurate and updated data.

Introduction

Sickle cell disease (SCD) is a hereditary blood disorder characterized by hemolytic anemia due to the propensity of the mutant hemoglobin to polymerize when deoxygenated, leading to deformation and ultimately hemolysis of red blood cells (RBCs).¹ Approximately two out of every 1000 births worldwide and more than 10 of every 1,000 births in Africa are affected by SCD.² SCD is characterized by the presence of hemoglobin S (HbS), a structural variant of normal adult hemoglobin, and occurs when mutated versions of the hemoglobin gene are inherited from both parents.³ Inheriting the HbS gene results in production of abnormal beta globin chains that polymerize when deoxygenated. The polymerization results in sickle shaped RBCs that have a markedly shortened life span, leading to moderate to severe anemia. Other effects of this process and the RBC abnormalities it engenders include increased RBC adhesion, activation of leukocytes and platelets, oxidative damage, and activation of coagulation and inflammatory pathways,⁴ all of which contribute to vaso-occlusion, as well as to complications such as strokes and acute chest syndrome.³

The severity of SCD varies greatly amongst individuals and correlates with a number of both hematologic and non-hematologic factors.^5,6 The rate of stroke and other complications in high-risk patients could effectively be reduced by more than 80% through the implementation of transfusion therapy.⁷ Chronic RBC transfusion limits the rate of stroke and consequently improves the life of SCD patients, but alloimmunization and severe iron overload have severe adverse effects.^7-10 Implementation of this valuable therapy appropriately and judiciously is therefore critical in prevention of both stroke and reducible complications. The goal of chronic RBC transfusion for patients with SCD is to: (i) increase the [Hb] (to 9-10 g/dL) to improve the oxygen-carrying capacity of blood; (ii) dilute sickle Hb (to HbS <30%) to decrease the multiple downstream effects listed above that contribute to stroke and vaso-occlusion; and (iii) increase tissue oxygenation to suppress the production of hemoglobin polymers.^7,8

Determining the need for transfusion therapy and the appropriate volume to transfuse for SCD patients is largely based on the pretransfusion Hb level, the pretransfusion %HbS, body weight, and clinical condition. Common methods used for monitoring the efficiency of transfusion therapy are Hb electrophoresis,¹¹ high performance liquid chromatography,¹² or isoelectric focusing.^13,14 These tests are performed at a high cost with long processing times. Thus, determining a SCD patient's need for transfusion therapy and the appropriate volume to transfuse is still largely based on the pretransfusion Hb level, body weight, and previous quantitative HbS measurements. Basing these decisions instead on the current %HbS could help tailor chronic transfusion for stroke prophylaxis, as well as acute transfusion for other SCD complications, more directly and effectively.¹⁵

Development of a rapid, cost effective, and point of care (POC) test used to quantify %HbS before, during and after transfusion therapy would ensure that current and accurate results are available to the healthcare provider when they are most valuable for decision-making. Several platforms have been developed to offer improved evaluations of SCD treatment.^16-18 We previously reported the development of a lateral flow immunoassay (LFIA) test¹⁵ to quantify and monitor HbS levels for patients going transfusion therapy as a SCD treatment. In this paper, we develop the technology of the POC quantitative HbS test and compare the LFIA test results with results from hemoglobin electrophoresis for 38 whole blood samples from SCD patients.

Protocol

This protocol follows institutional review board guidelines for ethical human research.

1. Preparation for Testing

1. Prepare test kit materials: collect Cartridge, Capillary Sampler, and Pretreatment Buffer Module, as well as materials needed for blood draw (K₂-EDTA vacutainer, alcohol swab, syringe, tourniquet, and bandage).
2. Turn on the reader via the power button located on the lower left side of the unit. Wait approximately 2 min for the software to boot and device to perform self-check.
3. When prompted, enter or barcode scan a User ID specific to the individual operator.
4. Press TEST on the reader touchscreen to be ready to run test.

2. Lot Verification Procedure

NOTE: A barcode that labels each Test Cartridge includes information about test name, calibration curve algorithm, lot number, and expiration date. If the current date exceeds the expiration date, the reader provides the user a warning that results may not be valid.

1. When new lot of reader or cartridges is received, perform Lot Verification to ensure proper performance of this or any IVD reagent. Run External Controls 1, 2, and 3 as in Section 3.
2. Record the output results. If the output results are in the range of what is indicated for the External Controls, the Test and Reader is ready for use.
   1. If External Controls do not report in the appropriate range, test the External Controls again. If the results are still not in the appropriate range, contact technical support.

3. Blood Sample Collection

1. Collect venipuncture whole blood samples from a patient who has the Hb genotype of HbSS and is to undergo transfusion treatment. Follow clinical protocols, but briefly:
   1. Select a large, firm vein, preferably in the antecubital fossa.
   2. To make the vein more prominent, apply a tourniquet and ask the patient to form a fist.
   3. Use 70% alcohol swabs to cover the whole area and ensure that the skin area is in contact with the disinfectant.
   4. Enter the vein at a 30° angle or less, and continue to introduce the 23 G (or size recommended by the institution's operating procedures for blood draw) needle along the vein at the easiest angle of entry.
   5. Once sufficient whole blood (~2 mL) has been collected in a K₂-EDTA anticoagulant vacutainer tube, release the tourniquet.
   6. Withdraw the needle gently and apply gentle pressure to the site with a clean gauze or dry cotton-wool ball.
   7. Immediately after withdrawal, invert the vacutainer tube 3 times.
   8. If the test will not be run within 4 h, store the vacutainer tube in 2-8 °C. Otherwise store the vacutainer tube at room temperature.

4. Testing Procedure

1. Collect a small volume of whole blood sample (5 µL) in the Capillary Sampler provided in the test kit.
2. Add the sample to the module containing proprietary PreTreatment Buffer immediately before testing.
3. Invert the module three times to cause cell lysis and to release hemoglobin.
4. Add 5 drops (100 µL) of the buffer-treated sample immediately onto the application site of the cartridge.
5. Insert the Test Cartridge into the reader when prompted. Slide the Test Cartridge in until it 'clicks' into place. The reader will automatically detect the barcode on the Test Cartridge with test function and calibration curve for the specific lot inserted.
6. Allow the test to run for 15 min with on-board timer in the reader for adequate detection and quantification of HbS. The %HbS will be shown on the screen. The output %HbS value is based on the inserted automated image analysis algorithm, which utilizes the colorimetric absorbance within specified areas of the test strip.

5. Clinical Application

1. Acquire general health information regarding the patient (i.e. age, gender, body weight, previous post-transfusion %HbS, other) and test for %HbS.
2. Apply transfusion (exchange transfusion or simple transfusion).¹⁹
3. Test %HbS before each blood pack is transfused into the patient. Keep monitoring and transfusing blood packs until the target %HbS in patient is achieved.
4. Once finished, record the post-transfusion %HbS to help physicians determine the time for next appointment.

Results

To enable the use of current and accurate results in the treatment of SCD patients, we have developed a POC test to quantify %HbS before, during, and after transfusion therapy. Our device applies the updated technology²⁰ of newly developed rabbit anti-human HbS monoclonal antibodies and a small quantitative reader to a highly-accessible LFIA format seen commonly in pregnancy tests and flu tests.

The HbS-L...

Discussion

The major goal of chronic RBC transfusion for SCD patients is to maintain a low %HbS (<30%) in order to reduce the rate of stroke and other severe complications.^7,21 Generally, the chronic exchange transfusion of 2-4 RBC units every 3-5 weeks is sufficient to keep the %HbS less than 30% and the [Hb] at 9-10 g/dL, thereby reducing the severe complications of SCD.^7,21 The transfusion frequency and volum...

Materials

Name	Company	Catalog Number	Comments
HbS test kit	BioMedomics	HLX020	Includes capillary samplers and pretreatment buffer modules
BioMedomics Quantitative Reader	BioMedomics	XJF-M
PreTreatment Buffer	BioMedomics	PT001	Contained within Pretreatment Module
K2-EDTA anticoagulant vacutainer tube	BD	367835	Please use as per Manufacturer instructions or your institution's standard operating procedures
Sterile Alcohol Prep Pads	Fisher Scientific	22-363-750	Please use as per Manufacturer instructions or your institution's standard operating procedures