We would like to thank the patients and parents for their continuous support; the caregivers for their dedication; Pr Bierling, director of the French Blood Bank in the Paris area, for his support of the collaborative work between our hospital and his team to develop the continuous manual exchange transfusion method; and the University Paris Diderot and the hospital Robert-Debr&#233; for their support.

The protocol follows the guidelines of the hospital ethics committee. There are 3 steps in the exchange sessions: patient preparation; initial isovolemic phlebotomy (if appropriate); and whole-blood exchange, consisting of several cycles, or continuous whole-blood phlebotomy, which is associated with the infusion of diluted PRBCs. Depending of the Hb level of the patient, a fourth step of intermediary isovolemic phlebotomy can be added midway through the exchange stage. At the start of the session, all of the necessary material (packed RBCs and 5% serum albumin) must be ready. Also, each step must be prepared in advance. Other than a precision scale and double venous access, no specific equipment is required. However, it is imperative to have constant medical supervision over the whole procedure, and even more so if the exchange volume is high.
1. Patient Installation
<ol>
	<li>Carry out laboratory tests prior to starting the exchange transfusion session. Perform them on the same day, less than 24 h before the session. Do not perform the tests before a physician checks the results of the following tests. 
	NOTE: The tests include a complete blood count, reticulocyte count, HbS rate measurement, indirect antiglobulin test (IAT), calcemia and electrolytes measurement, and liver function and coagulation tests, routinely performed by the hospital&#39;s hematology and biochemistry units.</li>
	<li>Have the physician perform a thorough and complete physical examination of the patient, and pay specific attention to hemodynamic parameters such as heart rate, blood pressure, respiratory rate, and oxygen saturation. Obtain a recent body weight. Maintain an oxygen saturation &#62;98% throughout the exchange session. To do so, administer patients with 1 L of oxygen via a nasal cannula while in supine position with the feet slightly raised.</li>
</ol>
2. Blood Product Preparation
<ol>
	<li>Calculate and obtain the initial phlebotomy volume (see Table 1). For example, if the initial Hb rate of the patient for the session is 10 g/dL, bleed 12 mL/kg of blood in 60 min. For a patient of 30 kg, bleed 360 mL of blood (these are appropriate volumes). 
	NOTE: An Hb rate of around 8 g/dL prior to the exchange transfusion must be reached because the initial phlebotomy volume is calculated based on the patient&#39;s Hb level (Table 1). 
	No prior phlebotomy is required if the Hb level is below 8.5 g/dL. The initial phlebotomy must not exceed 5 mL/kg of body weight in the case of patients who have suffered from a recent stroke (within the last 3 months).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Initial Hb level (g/dL)&#160;</td>
			<td>10</td>
			<td>9.5</td>
			<td>9</td>
			<td>8.5</td>
		</tr>
		<tr>
			<td>Volume to be bled (mL/kg)</td>
			<td>12</td>
			<td>10</td>
			<td>8</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Minimal duration of phlebotomy (min)</td>
			<td>60</td>
			<td>60</td>
			<td>45</td>
			<td>20</td>
		</tr>
	</tbody>
</table>
Table 1. Calculation of the Volume of the Initial Phlebotomy During Manual Exchange Transfusion. 
The initial phlebotomy volume is calculated based on the patient&#39;s initial Hb level, with the goal of reaching an Hb rate around 8 g/dL prior to the exchange transfusion. The duration of the initial bleeding depends on the volume of the phlebotomy. No prior phlebotomy is required if the Hb level is below 8.5 g/dL. The initial phlebotomy must not exceed 5 mL/kg of body weight in the case of patients who have suffered from a recent stroke.
<ol start="2">
	<li>Calculate the exchange volume; the total exchange volume is 35 - 45 mL/kg of body weight (e.g., if the patient is a child of 30 kg, the volume of the exchange transfusion will be around 1,200 mL). 
	NOTE: This volume is both the volume of blood that will bleed during the exchange step and the volume of diluted PRBCs that will be transfused to the patient during the exchange step. The final volume depends on the volume of the exchange transfusion, calculated with the body weight of the patient.</li>
	<li>Obtain the appropriate volume of 5% serum albumin solution (from the pharmacy) so that it is ready for use at the beginning of the session. Calculate the required volume: 50 - 100 mL to infuse prior to initiating the phlebotomy, plus the same volume during the initial phlebotomy to compensate for bleeding, plus 1/3 of the exchange volume to dilute the PRBCs.</li>
	<li>Obtain the appropriate volume of phenotypically matched PRBCs (i.e., 2/3 of the calculated exchange volume from the blood bank).</li>
	<li>Reduce the hematocrit of the PRBCs from 60% to 40% by diluting the PRBCs with 5% serum albumin. 
	NOTE: The dilution must be performed at the blood bank in a new blood bag. If this is not possible, use a 3-way tap to simultaneously transfuse the PRBCs and the 5% serum albumin and respect the flow proportions of 2/3 for the PRBCs and 1/3 for the albumin solution.</li>
</ol>
3. Patient Preparation
<ol>
	<li>Prepare two peripheral venous lines on two different limbs, one for the phlebotomy and one for the infusion of the albumin solution and PBRCs; the venous line for phlebotomy necessitates sufficient blood flow, and the one for infusion requires standard blood flow. Use a single venous access for infusion and phlebotomy with a 3-way tap if venous access is seriously limited.</li>
	<li>Administer 1 g of calcium per os to the patient before and after the exchange session; this prevents the occurrence of hypocalcemia due to the presence of a calcium chelating anticoagulant in the transfusion bags.</li>
</ol>
4. First Step of MET: Isovolemic Phlebotomy, if Appropriate
<ol>
	<li>Start the infusion of 5% albumin on one venous access. After infusing about 20 - 50 mL of albumin solution, begin phlebotomy on the second venous access.</li>
	<li>To perform the bleeding, install a peripheral intravenous access connected to an empty bleeding bag in the patient&#39;s arm. Place the bag below the level of the patient&#39;s bed. Observe the venous blood gradually fill the bleeding bag.
	<ol>
		<li>If the blood flow is too low, lift up the patient&#39;s bed (or lower the bleeding bag) in order to increase the height difference between the arm and the bleeding bag, thus increasing the blood flow. Optionally, in case of very low blood flow, have the nurse draw blood manually with a 50-mL syringe using a 3-way tap placed on the venous line. 
		NOTE: The flow of the phlebotomy must be the same as the flow of the infusion so as to strictly maintain the isovolemic balance.</li>
	</ol>
	</li>
	<li>Weigh the bleeding bag on a precision scale during the phlebotomy in order to adapt the infusion flow in real time to compensate for the volume bled. 
	NOTE: If no scale is available or if there is only a single venous access, bleed 20 mL of blood each time 20 mL of albumin is infused.</li>
	<li>At the end of the phlebotomy step, check the Hb levels using an Hb point-of-care test according to the manufacturer&#39;s instructions and make sure that it is around 8 g/dL.</li>
	<li>Monitor the patients every 5 min during the initial isovolemic phlebotomy step. Stop the phlebotomy if clinical changes relevant to the patient&#39;s age are observed.</li>
</ol>
5. Second Step of MET: Isovolemic Exchange Transfusion
<ol>
	<li>For safety reasons, start the transfusion of diluted PRBCs first. Transfuse the first 20 mL of blood and then start the phlebotomy. The planned total volume of the phlebotomy at this step is the same as the volume of the transfusion (35 - 45 mL/kg of body weight). 
	NOTE: The rate of the phlebotomy must be the same as the infusion rate of the diluted PRBCs, following the same method as in the previous step (i.e., weigh on a precision scale or alternate 20 mL cycles).</li>
	<li>Midway through the exchange step, check the Hb levels, as described in step 4.3. If the level is &#62; 9.5 g/dL, perform an additional round of phlebotomy. If not, continue the exchange transfusion.</li>
	<li>Monitor the patients every 15 min during the exchange transfusion step; a nurse must keep a close watch on the occurrence of any clinical and/or hemodynamic changes.</li>
</ol>
6. Additional Phlebotomy
<ol>
	<li>Perform an additional bleeding phlebotomy (see step 4.1) if the Hb levels midway through the exchange step are &#62; 9.5 g/dL, as there is a risk of reaching a too-high level of Hb at the end of the session. 
	NOTE: The volume of the additional phlebotomy step depends on the Hb level (Table 2). 
	Compensate with a 5% albumin infusion, as described above, in order to keep the additional phlebotomy isovolemic, just like in the other steps.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Midway Hb level (g/dL)&#160;</td>
			<td>10.5</td>
			<td>10</td>
			<td>9.5</td>
		</tr>
		<tr>
			<td>Volume to be bled (mL/kg)</td>
			<td>8</td>
			<td>6</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Minimal duration of phlebotomy (min)</td>
			<td>30</td>
			<td>20</td>
			<td>15</td>
		</tr>
	</tbody>
</table>
Table 2. Calculation of the Volume of the Intermediate Phlebotomy During Manual Exchange Transfusion. 
Perform an additional phlebotomy if the Hb level midway through the exchange step is higher than 9.5 g/dL, as there is a risk of reaching a too-high level of Hb at the end of the session. The duration of the initial bleeding still depends on the volume of the phlebotomy.
<ol start="2">
	<li>Just like the initial phlebotomy, monitor the patients every 5 min. Stop the phlebotomy immediately if clinical changes occur.</li>
	<li>After the additional phlebotomy, check the Hb levels using an Hb point-of-care test, and then continue the exchange procedure using the same method.</li>
</ol>
7. Final Laboratory Test
<ol>
	<li>At the end of the exchange transfusion, perform laboratory tests for Hb, HbS, and calcemia. Do not allow the patient to leave before the results of the laboratory tests (or at least the Hb levels) have been checked by a physician. 
	NOTE: Overall, while the duration of the procedure varies depending on the volume to bleed and to exchange, it lasts around 4 h, on average.</li>
</ol>

<ol>
	<li>Stuart, M. J., Nagel, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle-cell+disease.">Sickle-cell disease.</a> Lancet. 364 (9442), 1343-1360 (2004).</li><li>Aygun, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+transfusion+practices+for+prevention+of+primary+stroke+in+children+with+sickle+cell+anemia+and+abnormal+TCD+velocities.">Chronic transfusion practices for prevention of primary stroke in children with sickle cell anemia and abnormal TCD velocities.</a> Am J Hematol. 87 (4), 428-430 (2012).</li><li>Ohene-Frempong, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrovascular+accidents+in+sickle+cell+disease:+rates+and+risk+factors.">Cerebrovascular accidents in sickle cell disease: rates and risk factors.</a> Blood. 91 (1), 288-294 (1998).</li><li>Adams, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+a+first+stroke+by+transfusions+in+children+with+sickle+cell+anemia+and+abnormal+results+on+transcranial+Doppler+ultrasonography.">Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial Doppler ultrasonography.</a> N Engl J Med. 339 (1), 5-11 (1998).</li><li>Scothorn, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+recurrent+stroke+in+children+with+sickle+cell+disease+receiving+blood+transfusion+therapy+for+at+least+five+years+after+initial+stroke.">Risk of recurrent stroke in children with sickle cell disease receiving blood transfusion therapy for at least five years after initial stroke.</a> J Pediatr. 140 (3), 348-354 (2002).</li><li>Koehl, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+automated+erythrocytapheresis+versus+manual+exchange+transfusion+to+treat+cerebral+macrovasculopathy+in+sickle+cell+anemia.">Comparison of automated erythrocytapheresis versus manual exchange transfusion to treat cerebral macrovasculopathy in sickle cell anemia.</a> Transfusion. 56 (5), 1121-1128 (2016).</li><li>Kim, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocytapheresis+therapy+to+reduce+iron+overload+in+chronically+transfused+patients+with+sickle+cell+disease.">Erythrocytapheresis therapy to reduce iron overload in chronically transfused patients with sickle cell disease.</a> Blood. 83 (4), 1136-1142 (1994).</li><li>Adamkiewicz, T. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+ferritin+level+changes+in+children+with+sickle+cell+disease+on+chronic+blood+transfusion+are+nonlinear+and+are+associated+with+iron+load+and+liver+injury.">Serum ferritin level changes in children with sickle cell disease on chronic blood transfusion are nonlinear and are associated with iron load and liver injury.</a> Blood. 114 (21), 4632-4638 (2009).</li><li>Raghupathy, R., Manwani, D., Little, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+overload+in+sickle+cell+disease.">Iron overload in sickle cell disease.</a> Adv Hematol. , (2010).</li><li>Porter, J. B., Evangeli, M., El-Beshlawy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+of+adherence+and+persistence+with+iron+chelation+therapy.">Challenges of adherence and persistence with iron chelation therapy.</a> Int J Hematol. 94 (5), 453-460 (2011).</li></ol>

The authors have nothing to disclose.

The risk associated with this procedure is an unexpected misbalance between the phlebotomy and the transfusion, which can have dangerous consequences. A rapid depletion will lead to hypovolemia and acute anemia, while an excess transfusion without bleeding will lead to a dangerous increase in blood viscosity. In both cases, SCA patients could suffer from vaso-occlusive complications, as well as strokes. For this reason, one nurse must be dedicated to each patient and stay at his bedside during the whole procedure. There ...

A single point mutation in the &#946;-globin gene is responsible for the production of abnormal hemoglobin (hemoglobin S, HbS). This causes sickle cell anemia (SCA), one of the most common diseases worldwide1. SCA patients&#39; acute symptoms and some chronic complications can be treated by the transfusion of packed red blood cells (PRBCs). Indeed, the transfusion of normal RBCs corrects the anemia while diluting the sickle RBCs. As a result, it can increase the oxygen transport capacity while decreasing hemolysis and vaso-occlusive events. To avoid chronic complications or to treat patients with acute complications, transfusion combined with the depletion of sickle RBCs, either by phlebotomy or by erythrapheresis, is an effective way to limit the dangerous increase in hemoglobin and blood viscosity while reducing the number of circulating SS RBCs2.
One of the main causes of psychomotor handicaps and neurocognitive deficiencies in children with SCA3 is cerebral vasculopathy, a devastating complication of this disease. In SCA children with abnormally high velocities on transcranial Doppler, chronic transfusions are effective in preventing the occurrence of the first stroke4. To reduce the risk of recurrence in patients that have already suffered from an ischemic stroke, transfusion therapy is the most adequate method5. In the case of chronic therapy, RBC exchange transfusion is better than simple RBC transfusion, as it removes sickle cells and adds normal cells while reducing blood viscosity and limiting iron overload. Nonetheless, simple RBC transfusion is still widely used as a treatment for cerebral macro-vasculopathy. While it rapidly leads to iron overload4, this choice is often made because it is technically simple and maximizes the number of patients in transfusion care. Indeed, even if erythrapheresis has been reported to be the most efficient method for the chronic transfusion of SCA patients, it cannot be implemented everywhere; it is not suitable for all patients, especially young children; and it necessitates specific and expensive equipment.
For more than 20 years now, we have been treating SCA children demonstrating cerebral vasculopathy and who were temporarily ineligible for erythrapheresis with a continuous manual exchange transfusion (MET) method. In 2016, our team published a follow-up of patients that had undergone continuous manual transfusion for several years, showing that our method is associated with a satisfactory HbS decrease, efficient stroke prevention, and a limitation of iron overload comparable to that of erythrapheresis6. A session of MET can be carried out in any hospital environment without specific apparatuses and by using the same volume needed for erythrapheresis. A notable advantage of this technique is that it could help prevent, or at least diminish, side effects (particularly iron overload) linked to repeated transfusions in patients who are not able to undergo erythrapheresis. The aim of this article is to describe, step by step, how to perform a session of continuous MET in order to allow the medical centers that do not have any apheresis machines, or that have patients who are not eligible for erythrapheresis, to use this method for their SCD patients, especially children.

<table><tbody><tr><td>Precision scale</td><td></td><td></td><td></td></tr><tr><td>Cannula (x2)</td><td>Macopharma</td><td></td><td></td></tr><tr><td>Transfusion tubing (x2)</td><td>Macopharma</td><td></td><td></td></tr><tr><td>Bleeding bag (x4)</td><td>Macopharma</td><td></td><td></td></tr><tr><td>3 Way tap</td><td></td><td></td><td></td></tr><tr><td>Syringe (x4)</td><td></td><td></td><td></td></tr><tr><td>Hemoglobin test</td><td>HemoCue</td><td>Hb 201&lt;sup&gt;+&lt;/sup&gt;&amp;nbsp;System</td><td></td></tr></tbody></table>

continuous manual exchange transfusion for patients with sickle cell disease: an efficient method to avoid iron overload

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. Repeated transfusions of packed red blood cells (PRBCs) is currently the simplest and most used technique for chronic transfusion programs. However, iron overload is one of the major side effects of this therapy. More developed methods exist, notably the apheresis of RBC (erythrapheresis), which is currently the safest and most efficient method. However, it is costly, complicated, and cannot be implemented everywhere, nor is it suitable for all patients. Manual exchange transfusions combine one or more manual phlebotomies with a PRBC transfusion.
At the Reference Center of Sickle Cell Disease, we set up a continuous method of manual exchange transfusion that is feasible for all hospital settings, demands no specific equipment, and is widely applicable. In terms of HbS decrease, stroke prevention, and iron overload prevention, this method showed comparable efficiency to erythrapheresis. In cases where erythrapheresis is not available, this method can be a good alternative for patients and care centers.

Here, we will compare the safety, cost, and efficiency of the MET method with erythrapheresis6, which is the most effective method to decrease the percentage of HbS in SCD patients. To do so, we recorded 1,353 transfusion exchange sessions in the Reference Center of SCD, including 333 sessions of AET and 1,020 sessions of MET, all in SCD children suffering from cerebral vasculopathy and/or strokes. For patients, we chose MET in children with a body weight under 25 ...

Watch this Scientific Journal Video about Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload at JoVE.com

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment.

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. ...

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Research

JoVE Journal

Medicine

19.4K Views.  Robert Debré Hospital, AP-HP.  We hebben een werkwijze voor continue handmatige uitwisselingstransfusie beschreven voor de behandeling van sikkelcelziekte bij patiënten. Deze veilige protocol is ontworpen om effectief beperken ijzerstapeling bij patiënten die behoefte hebben aan chronische transfusies en uitgebreid kan worden gebruikt zonder speciale apparatuur. 

Video: Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Developmental Biology

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Immunology and Infection

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Bioengineering

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

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 &#60;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 &#177;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.

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.

Sickle cell disease (SCD) causes many severe health complications, including anemia, stroke, and acute chest syndrome. Red blood cell transfusion is the ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

Biology

POC Method and Integrated Decision Support Tool for Estimating Anemia

A Point-of-Care Method with Integrated Decision Support Tool to Estimate Anemia at Population Level

Robust point-of-care methods are required to estimate anemia at the population level. The accurate methods are lab-based and cannot be used at the point of care. To address this caveat, a novel method based on pooled capillary blood and a portable autoanalyzer was developed for the estimation of Hb. Additionally, custom software was developed for near-real-time integration of the Hb values from the auto analyzer to the server. Moreover, a decision support tool that can immediately categorize the participants into different stages of anemia was developed. The decision support tool was designed based on the World Health Organization (WHO) cut-off for anemia at the population level and was available for all age and gender groups. This simple and user-friendly tool could&#160;easily be used by front-line health workers who have limited technical skills. Overall, the method developed could&#160;be used at the point of care and is accurate. This high-throughput method could&#160;be used for screening anemia at the population level for all age and gender groups.

Author Spotlight: Developing a Point-of-Care Hemoglobin Estimation Method for Anemia Management

An accurate hemoglobin estimation method is lacking at the point of care and may hinder population-based programs for treating anemia. Therefore, we developed a point-of-care method based on pooled capillary blood and an auto-analyzer integrated into a custom software application to categorize the hemoglobin values into different grades of anemia.

Robust point-of-care methods are required to estimate anemia at the population level. The accurate methods are lab-based and cannot be used at the point ...

Modeling Erythropoiesis Using HSPCs from Cord Blood and Bone Marrow

A Comprehensive Pipeline to Assess the Efficiency of Human Erythropoiesis In Vitro and Ex Vivo

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 &plusmn; 20 billion cells per day), is crucial for maintaining individual health. Any disruption in this pathway can have significant consequences, leading to health issues. According to the World Health Organization, an estimated 25% of the global population presents symptoms of anemia. This protocol describes how to generate human erythroid cells both in vitro using hematopoietic stem and progenitor cells (HSPCs) from sources such as umbilical cord blood (UCB) or blood taken from healthy donors and ex vivo with HSPCs isolated from patients' bone marrow. Using genetic approach, genes of interest can be modulated in HSPCs, and their impact on erythropoiesis can be monitored at various stages of the differentiation process. This method allows for the screening of compounds perturbing, enhancing, or rescuing the capacity of HSPCs to differentiate into mature erythroid cells and to investigate the role of genes of interest during the erythroid differentiation process.

Author Spotlight: Advancing Erythropoiesis Research - A Simplified Pipeline for Assessing Hematopoietic Stem Cell Function in Myelodysplastic Syndromes

Modeling erythropoiesis provides a valuable chance to comprehend the biological significance of actors in this process and to evaluate novel therapeutic approaches that may alleviate differentiation defects. Here, we describe a simple and reliable method to efficiently differentiate CD34+ hematopoietic stem and progenitor cells ex vivo.

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 &plusmn; 20 ...

Blood Transfusion and Agglutination

Blood transfusion is a therapeutic measure to restore the blood volume after extensive blood loss due to an accident or a medical procedure. Blood transfusion involves drawing a certain amount of blood from a suitable donor and infusing it into the recipient.
<h4>History</h4>
The history of blood transfusion dates back to the 17th century, when early attempts were made in animals. In 1818 James Blundell, a British doctor, performed the first successful human blood transfusion. Later in 1900, Karl Landsteiner discovered A, B, and O blood types and paved the way for scientific blood transfusion techniques.
<h4>Blood fractions</h4>
While whole blood is usually used during the transfusion event, however, based on the requirement, different blood components can also be used separately. For example, platelet fraction of donor blood is used to treat excessive bleeding caused by platelet deficiency. The immunoglobulins in the plasma are transfused to protect patients against viral infections.
<h4>Immune-compatible blood</h4>
Before the blood transfusion, several checks are run to ensure that the blood is safe from pathogens such as HBV, HCV, HIV, and HTLV-I/II and does not induce an immune reaction in the recipient&#8217;s body. For example, type-A blood cannot be given to an acceptor with type-B, or O, because the acceptors&#8217; immune system would produce antibodies against incoming antigen-A, leading to a blood agglutination reaction in the host body.
Due to the lack of antigens, blood type-O does not elicit immune reactions with other blood types. Therefore, type-O blood can be given to patients with all other blood types. Conversely, a person with blood type-AB has no antibodies to either antigen A or B. Similarly, if an Rh-negative mother has a fetus with Rh-positive blood, she may produce antibodies against the fetus blood, resulting in the fatal hemolytic disease in the newborn.

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

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