When recombinant proteins can replace rare red cells in immunohematology workups

Many blood group antigens are present in >90% of people¹ worldwide or in large populations. They are high-prevalence antigens, often with frequencies >99%.^2–5 They are also known as high-incidence^3,6–8 or high-frequency antigens,^9–11 while the term public antigens¹² is not used anymore. Low-prevalence blood group antigens occur in <1% of people.¹ Pairs of corresponding high- and low-prevalence antigens are antithetical antigens.¹³ Individuals whose red cells completely lack the protein or carbohydrate, often bound to lipids, carrying blood group antigens can develop antibodies to high-prevalence antigens when challenged with normal red cells through blood transfusion or pregnancy. Alternatively, individuals who are homozygous or heterozygous for alleles expressing a low-prevalence antigen, for example, caused by an amino acid substitution in the protein, can develop an antibody to the original protein expressing the high-prevalence antigen.¹⁴

Many antibodies to high-prevalence antigens are clinically significant.^15–17 These antibodies are difficult for routine laboratories to investigate due to a dearth of red cells that are negative for high-prevalence antigens.^15,18 In this method note, we advocate the application of recombinant proteins in routine immunohematology workups to help identify antibodies against high-prevalence antigens.

1 |. RECOMBINANT BLOOD GROUP PROTEINS

Artificially produced proteins are called recombinant proteins, when they are expressed in vector systems using the recombinant DNA technology in cells.¹⁹ The technology, established in the early 1970s,²⁰ was first applied to therapeutic protein production in the late 1970s²¹ (human insulin²²) and early 1980s²³ (tissue plasminogen activator^24,25) and became widely available in the 1990s.¹⁹

Recombinant blood group proteins (rBGPs) expressing functional blood group antigens can react with human antisera in conventional hemagglutination assays. rBGPs have occasionally been used in defining new blood group antigens, generally in conjunction with rare red cells. Molds and Rowe, in 1996, were the first to describe the soluble form of a protein to neutralize antibodies against distinct high-prevalence blood group antigens.²⁶ They applied a soluble form of the Complement Receptor 1 (CR1) protein in a hemagglutination inhibition assay and neutralized antibodies against high-prevalence antigens of the Knops blood group system.²⁶

Red cells negative for high-prevalence antigens are often in short supply. No Immunohematology Reference Laboratory (IRL) may have all of them available. Because these red cells are lacking or scarce, many studies began utilizing rBGPs.^27–40 Also, rBGPs are easy to store frozen or lyophilized, quite different from the complex shipping and freezing requirements of red cells.⁴¹ The expression of rBGP in eukaryotic systems is often preferred over expression vectors in prokaryotic systems, although they are easier and cheaper,⁴² because appropriate posttranslational modifications are required for the formation of three-dimensional epitope structure.⁴³

2 |. rBGPs AND ANTIBODY CHARACTERIZATION

Since 1996, multiple studies have exploited soluble or cell membrane-bound recombinant proteins to screen human sera for blood-group-specific antibodies (Table 1).^{27,29,32,44–95} There are 190 red cell antigens classified as high-prevalence by the ISBT (see web resources).⁹⁶ To identify antibody specificities against all high-prevalence antigens, up to 190 distinct red cells lacking high-prevalence antigens or lacking the whole carrier protein would be needed. When available at all, such red cells may not be blood group O and then be incompatible with the non-O patients.

TABLE 1.

Blood group systems and their antigens whose antibodies have been inhibited by soluble recombinant proteins

					Comment
Blood group system	Symbol	ISBT number	Antigen	Prevalence	Reference	Commercial availabilitya
MNS	MNS	002	M	Intermediate	44	No
			Vw	Low	44	No
			Hut	Low	44	No
Lutheran	LU	005	Lua	Intermediate	32,46–48	Yes
			Lub	High	45–47,49–51	Yes
			Lu3	High	46	No
			Lu4	High	51	Yes
			Lu5	High	51	Yes
			Lu6	High	51	Yes
			Lu7	High	51	No
			Lu8	High	32	Yes
			Lu12	High	51	Yes
			Lu13	High	51	Yes
			Aub	Intermediate	51	No
			Lu20	High	51	Yes
			Lu28	High	52	Yes
			Lu29	High	53	Yes
Kell	KEL	006	K	Intermediate	45,46,54–56	Yes
			k	High	45,46,54,55,57,58	Yes
			Kpa	Low	56,57	No
			Kpb	High	45,46,54,57	Yes
			Ku	High	57,58	No
			Jsa	Low	56	Yes
			Jsb	High	45,46,54,57,58	No
			K14	High	58,59	Yes
Duffy	FY	008	Fya	Intermediate	45,46,57,60–62	Yes
			Fyb	Intermediate	45,46,57,61,62	Yes
			Fy6	High	60	Yes
Kidd	JK	009	Jkb	Intermediate	63	No
Diego	DI	010	Wrb	High	64	No
Cartwright	YT	011	Yta	High	32	Yes
			YTEG	High	65	Yes
			YTLI	High	66	Yes
			YTOT	High	66	Yes
Xg	XG	012	Xga	Intermediate	67	Yes
			CD99	High	67,68	No
Scianna	SC	013	Sc1	High	29	Yes
			Sc3	High	69	Yes
			STAR	High	29	Yes
			SCAR	High	70	Yes
Dombrock	DO	014	Doa	Intermediate	32,71	Yes
			Dob	Intermediate	32,72	Yes
			Gya	High	71,72	No
			Hy	High	71	Yes
			Joa	High	71	Yes
Colton	CO	015	Not specified	Not specified	73	NA
Landsteiner-Wiener	LW	016	LWa	High	74	Yes
			LWb	Low	74	No
Chido/Rodgers	CH/RG	017	Not specified	High	32	Yesb
Kx	XK	019	Kx	High	59	NA
Gerbich	GE	020	Ge2	High	27	No
			Ge3	High	27,75	No
			Ge4	High	27	No
			Lsa	Low	27	No
Cromer	CROM	021	Cra	High	32,76,77	Yes
			Tca	High	76,77	Yes
			Dra	High	76	Yes
			IFC	High	76	Yes
			WESb	High	32	Yes
			UMC	High	76	Yes
			CORS	High	78	Yes
Knops	KN	022	Kna	High	26,57,79,80	Yes
			McCa	High	5,57,80	Yes
			Sla	High	26,80	Yes
			Yka	High	5,57,80	Yes
			DACY	High	81	Yes
			YCAD	Intermediate	81	Yes
			Not specified	Not specified	32,57,82	NA
Indian	IN	023	Inb	High	83	Yes
			INSL	High	84	Yes
Ok	OK	024	Not specified	Not specified	85,86	NA
John Milton Hagen	JMH	026	JMH	High	32,87,88,90,91	Yes
			JMHK	High	88	Yes
			JMHL	High	88	Yes
			JMHG	High	88	Yes
			JMHM	High	88	Yes
			JMHQ	High	89	Yes
			JMHN	High	92	Yes
			JMHA	High	93	Yes
CD59	CD59	035	CD59.1	High	94	Yes

Abbreviations: ISBT, International Society of Blood Transfusion; NA, not applicable.

^aList of commercially available rBGPs with corresponding antigens (see Web resources).

^bChido/Rodgers antigens targeted: Ch1 and Rg1.

A small number of readily available rBGPs may suffice to inhibit antibodies against high-prevalence blood group antigens, once the rBGPs are made available and their functionality has been shown and comprehensively investigated. Besides the established rBGPs in the 21 blood group systems (Table 1), this may in the future also apply to the remaining 13 protein-based blood group systems (Table 2). We propose that laboratories keep a panel of rare red cells along with a panel of rBGPs. However, rBGPs need to be developed for most of the low-prevalence antigens as currently commercially available rBGPs are predominantly directed against high-prevalence antigens (see web resources).

TABLE 2.

Blood group systems whose antibodies could potentially be inhibited by soluble recombinant proteins^a

Blood group system	Symbol	ISBT number
Rh	RH	004
Raph	RAPH	025
Gill	GIL	029
Rh-associated glycoproteinb	RHAG	030
JR	JR	032
LAN	LAN	033
Vel	VEL	034
Augustine	AUG	036
KANNO	KANNO	037
CTL2	CTL2	039
PEL	PEL	040
MAM	MAM	041
ABCC1	ABCC1	043

Abbreviations: ISBT, International Society of Blood Transfusion.

^aAntibodies of these blood group systems may be feasible targets for inhibition by different reagents, such as recombinant blood group proteins (rBGPs), peptides, mimotopes, or aptamers. However, such inhibition has not been shown for any of them. Some of the reagents may be commercially available.

^bKg antibody inhibited by the RhAG protein expressed on the surface of Chinese Hamster Ovary cells.⁹⁵

3 |. RECOMBINANT PROTEINS AND NEW BLOOD GROUP SYSTEMS

More red cell membrane proteins than blood group systems are known^97–99 to be of clinical relevance.¹⁰⁰ Some will become defined as new blood group systems in the future, such as the recently discovered ABCC1 blood group system (Table 2).^100,101 Soluble forms of red cell membrane proteins obtained through recombinant expression systems can act as antigens to inhibit novel antibodies contributing to the identification of new blood group systems. Once defined as a new system, the proteins can be listed as rBGPs.

4 |. rBGPs AND NOVEL ANTIGENS IN KNOWN BLOOD GROUP SYSTEMS

To confirm a new high-prevalence antigen actually belonged to the Scianna blood group system, we recently applied a soluble recombinant ERMAP protein.⁷⁰ The ERMAP protein inhibited the antibody in our study, which allowed us to define the antibody as being directed against a hitherto unknown high-prevalence Scianna antigen, dubbed SCAR (SC8).⁷⁰ The standard approach was using red cells with rare null phenotypes (null cells)^102–104 to define new blood group systems and new high-prevalence antigens. Unless alternatives are available, null cells are needed because ISBT guidelines¹ for establishment of new blood group systems or new antigens in an established system require demonstration of antigen expression specifically in erythroid cells.

The ISBT Working Party on Red Cell Immunogenetics and Blood Group Terminology has begun accepting evidence from rBGPs alone,^{65,66,70,81,84} without testing against red cells lacking the protein or antigen, to satisfy the requirements¹ for defining novel blood group antigens. The first example was the YTEG antigen of the YT blood group system in 2017,⁶⁵ followed by the YT antigens YTLI and YTOT⁶⁶ and the Indian antigen INSL⁸⁴ in 2018. SCAR⁷⁰ in the Scianna blood group system¹⁰⁵ and DACY and YCAD⁸¹ in the Knops blood group system¹⁰⁶ were accepted in 2020 based on rBGP evidence without testing against red cells lacking the relevant blood group protein.

In many blood group systems, carriers of the null phenotype are rare.¹⁰² In a few blood group systems, notably YT,¹⁰⁷ the null phenotype may be incompatible with life or at least no living carrier of null cells has ever been found. Hence for YT, the definition of new blood group antigens^108,109 was by necessity without red cells lacking the AChE protein. For Scianna and Indian, null cells albeit very rare are known,^110,111 and the description of the new antigens SCAR and INSL was by choice without red cells lacking the ERMAP or CD44 proteins. The serologic KN “null” phenotype, known as Helgeson phenotype, is not a true null phenotype as it expresses low amounts of KN antigens.¹¹²

Antibodies against high-prevalence antigens occur in individuals who are homozygous for exquisitely rare alleles. Hence, a convincing biostatistical argument can be made to obviate the need for family or experimental studies, if null cells or rBGPs are used. Antibodies against low-prevalence antigens typically require family studies to show inheritance. If unavailable, technically advanced expression studies in human erythroid systems used to be the only option left. Today, rBGPs may be the technically easier way to express a low-prevalence antigen and prove an antibody’s specificity. For all new antigens, several lines of evidence are sought and rBGPs are just another, yet readily available, line of evidence. The approach of using rBGPs may have a wider application and would enable more laboratories to participate in the search and definition of new blood group antigens.

5 |. rBGPs AND PROTEIN BLOOD GROUP SYSTEMS

Antibodies against any antigen of the 34 protein-based blood group systems can be inhibited. Depending on the structure of the protein in the red cell membrane, suitable rBGPs for many blood group systems have been developed (Table 1). In case of proteins spanning the red cell membrane, soluble recombinant polypeptides representative for the extracellular regions are required. Blood group systems with glycosylphosphatidylinositol (GPI)-anchored proteins¹¹³ can be artificially expressed.⁹⁴ Another source for this type of protein is its cleavage from the GPI-anchor on red cells by using a specific bacterial phospholipase, phosphatidylinositol-phospholipase C (PI-PLC).^114,115

6 |. rBGPs AND NON-PROTEIN BLOOD GROUP SYSTEMS

rBGPs, as proteins, can obviously not inhibit antibodies against the sugar antigens in the eight carbohydrate-based blood group systems^116,117 or the one recently described blood group system constituted by the GPI anchor itself.^118,119 It has been known long before 1996 that most of these antibodies can, however, be inhibited by biologicals. They include soluble substances present in human bio-fluids (saliva, urine, milk, and plasma), hydatid cyst fluid, pigeon egg albumin, or guinea pig urine.⁴⁰

7 |. rBGP ALTERNATIVES

Besides somewhat ill-defined biologicals,⁴⁰ there are well-defined rBGP alternatives, which can serve as tools in immunohematology today and function similar to rBGPs. Short peptides called mimotopes^120,121 or peptide aptamers^122,123 are 5–20 amino acid residues long fragments that can be used as soluble agents to neutralize blood group antibodies in hemagglutination inhibition assays. The peptides can structurally mimic both carbohydrate and protein epitopes as shown for Lewis,¹²⁴ and ABO¹²⁵ as well as Rh¹²⁶ and Duffy blood group systems.^127,128 These rBGP alternatives are chemically synthesized, inexpensive peptides requiring neither recombinant DNA technology nor prokaryotic and eukaryotic cells to produce.

8 |. SUMMARY

rBGPs are feasible substitutes for red cells with rare null phenotypes (null cells) and can contribute to identify antibodies against high-prevalence antigens. However, several limitations restrict the widespread use of rBGPs in the identification of new blood group systems and new antigens in known systems. These limitations include the efficient production of rBGPs in soluble form with precise folding, the lack of rBGPs for many blood group systems, and of course cost. We still expect that this methodology can often replace the use of null cells or protein expression studies in human erythroid cells, which have hitherto been considered the gold standard for proof of novel blood group antigens and systems.¹ This approach has recently been accepted by the ISBT Working Party on Red Cell Immunogenetics and Blood Group Terminology. Panels of commercially available rBGPs could be developed, which in different combinations would provide handy sets of reagents for use in routine blood group serology, antibody screening, and antibody identification.³⁹

Abbreviations:

CR1

Complement Receptor 1

IRL

Immunohematology Reference Laboratory

ISBT

International Society of Blood Transfusion

rBGPs

Recombinant blood group proteins