ALPF Medical Research

Blood Donors with McLeod Phenotype

More than 300 antigens are recognized on the red cell surface and can be classified into 4 separate categories; these are 29 genetically determined systems, 6 collections of related antigens, and the series of lo w incidence (700 series) and high incidence

Y. Tani

Japanese Red Cross Osaka Blood Center, 2-4-43, Morinomiya Joto-ku, Osaka, 536-8505, Japan, [email protected]

(901 series) antigens [2]. Among the 29 systems (Table 1), red cells which lack XK protein on the cell surface are called McLeod phenotype. McLeod phenotype is one of the rare blood cell types, defined as those that occur at a frequency of 1:1,000 or less, and was first reported by Allen et al. in 1961 [1].

In Japan, we have screened donors for rare cell types since 1987 using monoclonal antibodies (MAbs) which we have developed. Our screening strategy for McLeod phenotype is shown in Fig. 1. We do not have good MAbs against XK protein, but Kell glycoprotein is covalently linked at Cys 72 to Cys 347 of the XK protein in the red cell membrane (Fig. 2) [8] and it is well known that Kell antigen expression is markedly reduced in the absence of normal XK protein. We screen red cells by agglutination methods in an automated system such as Olympus PK7200

Table 1 The blood group systems, the genes that encode them, and their chromosomal location (ISBT, 2004) [2]

System Chromosomal CD

No. System name symbol Gene name(s) location numbers

Table 1 The blood group systems, the genes that encode them, and their chromosomal location (ISBT, 2004) [2]

System Chromosomal CD

No. System name symbol Gene name(s) location numbers

001	ABO	ABO	ABO	9q34.2
002	MNS	MNS	GYPA, GYPB, GYPE	4q31.21	CD235
003	P	P1		22q11.2-qter
004	Rh	RH	RHD, RHCE	1p36.11	CD240
005	Lutheran	LU	LU	19q13.32	CD239
006	Kell	KEL	KEL	7q34	CD238
007	Lewis	LE	FUT3	19p13.3
008	Duffy	FY	FY	1q23.2	CD234
009	Kidd	JK	SLC14A1	18q12.3
010	Diego	DI	SLC4A1	17q21.31	CD233
011	Yt	YT	ACHE	7q22.1
012	Xg	XG	XG, MIC2	Xp22.32, Yp11.3	CD99b
013	Scianna	SC	ERMAP	1p34.2
014	Dombrock	DO	DO	12p12.3
015	Colton	CO	AQP1	7p14.3
016	Landsteiner-Wiener	LW	ICAM4	19p13.2	CD242
017	Chido/Rodgers	CH/RG	C4A, C4B	6p21.3
018	Hh	H	FUT1	19q13.33	CD173
019	Kx	XK	XK	Xp21.1
020	Gerbich	GE	GYPC	2q14.3	CD236
021	Cromer	CROM	DAF	1q32.2	CD55
022	Knops	KN	CR1	1q32.2	CD35
023	Indian	IN	CD44	11p13	CD44
024	Ok	OK	BSG	19p13.3	CD147
025	Raph	RAPH	CD151	11p15.5	CD151
026	John Milton Hagen	JMH	SEMA7A	15q24.1	CD108
027	I	I	GCNT2	6p24.2
028	Globoside	GLOB	B3GALT3	3q26.1
029	GIL	GIL	AQP3	9p13.3

Kell antigen testing on the Olympus PK7200 or Toraymac GR

^ using monoclonal anti-k, anti-Ku or anti-K14 by Bromelin

Weak or Negative i

Retest for indirect antiglobulin test with polyclonal and other monoclonal anti-Kell

Weak Negative

McLeod or Kmod Ko

Flow cytometric analysis !

Scanning electron microscopy ;

Indirect antiglobulin test with anti-Kx !

XK gene analysis ;

Fig. 1 Flow chart of screening of red cells for McLeod phenotype

Fig. 2 Kell/XK complex (modified from Lee et al. [8]). Kell antigen is 93 kDa type II glycoprotein consisted of 732 amino acid polypeptides and XK is 37 kDa, 444 amino acid polypeptides. They are covalently linked by a disulphide bond. (C: cysteine)

or Toraymac GR using anti-Kell MAbs (anti-k, anti-Ku and anti-K14 etc.). In the western regions of Japan from 1987 to 2005, a total of 16,160,714 donations of red blood cells (including repeat donors) were screened using mouse monoclonal anti-k (OSK5), anti-Ku (OSK32) and anti-K14 (OSK25).

We found 182 cell donations with McLeod phenotype or Kmod and 286 with Ko. Kmod red cells express Kell antigens weakly, and Ko express no Kell antigens. XK protein expression is intact or even increased in these two phenotypes as opposed to the McLeod phenotype. To identify McLeod phenotype after screening, we examine the expression level of Kell antigens by flow cytometry (Fig. 3); cell morphology (acanthocytes) by scanning electron microscopy (Fig. 4); serum creatine phosphokinase (CPK) and haptoglobin (Hp); red cells of family members (especially mothers), and the XK gene.

Although we have not examined all 182 donors, we have identified 3 donors with McLeod phenotype (2 subsequently developed McLeod syndrome [13, 16], 2 with McLeod-like phenotype (McLeod-like red cells show the same serological reactions and acanthocytes as McLeod except they weakly react with anti-Kx) [10], and more than 50 with Kmod. Nine donors are currently registered as McLeod phenotype in the whole of Japan [3, 11, 14]. Their CPK is usually high and Hp usually low (Table 2). Of note, one donor (M-1) developed McLeod syndrome 15 years after we identified him, although his CPK was within the normal range at initial screening.

Red cells	Fluorescence/cell
Ko	4.5
Normal	156.0
McLeod	12.9
Mother	55.6 two peaks

Fig. 3 Kell antigen expression in McLeod male, female carrier (his mother) and Ko donors. Flow cytometry using anti-Kpb (OSK36) shows that Kell antigens are weak on red blood cells from the McLeod male and that the female carrier has a mixed population (two peaks) because XK is subject to X-chromosome inactivation

Fig. 3 Kell antigen expression in McLeod male, female carrier (his mother) and Ko donors. Flow cytometry using anti-Kpb (OSK36) shows that Kell antigens are weak on red blood cells from the McLeod male and that the female carrier has a mixed population (two peaks) because XK is subject to X-chromosome inactivation

Noun alConüDl

Fig. 4 Scanning electron micrographs of acanthocytes in McLeod male and female carrier (mother). A lower percentage of acanthocytes is observed in the female carrier (JSM-5410, x1,500)

Noun alConüDl

Fig. 4 Scanning electron micrographs of acanthocytes in McLeod male and female carrier (mother). A lower percentage of acanthocytes is observed in the female carrier (JSM-5410, x1,500)

Table 2 CPK and haptoglobin	in McLeod phenotype donors
	Age detected
Donor No.	(report)	CPK (U/L)	Hp (mg/dL)	References
McLeod M-1a	21 (1989)	56	n.t.	[13, 15]
M-2	45 (1994)	921	<10	[3]
M-3	31 (1995)	683	<10	[11]
M-4	29 (1995)	692	51	[14]
M-5	34 (2000)	1,937	<10	[14]
M-6	20 (2000)	282	152	[14]
M-7	27 (2000)	439	<10	[14]
M-8	? (2000)	862	<10	[14]
McLeod-likeb M-9	21 (1989)	382	<11.5	[10]
Normal		57-197	41-341

CPK creatine phosphokinase, Hp haptoglobin a Developed McLeod syndrome at 36 years old b McLeod-like: the same laboratory findings as McLeod apart from weak reaction with anti-Kx

Table 3 Differences among McLeod syndrome, chorea-acanthocytosis and non-acanthocytic chorea

	Antigen expression		RBC	Possible antibodies
	Kell	Kxa	Morphology	Produced	CPK
Normal Kell	N	N	N	N	N
Ko (Kell null)	(-)	Increased	N or elliptocyte	Anti-Ku	N
Kmod	Reduced	Increased	N	Anti-Ku-like	N
McLeod syndrome	Reduced	(-)	Acanthocyte	Anti-Kx, Km	N or High
McLeod carriers	Reduced to Nb	(-) to N	Acanthocyte	N	?
Chorea-acanthocytosis	N	N	Acanthocyte	N	High
Chorea	N	N	N	N	High
(non-acanthocytic)

CPK creatine phosphokinase, N normal a Anti-Kx + Km kindly gifted by Marion Reid, New York Blood Center b Flow cytometry: two peaks

CPK creatine phosphokinase, N normal a Anti-Kx + Km kindly gifted by Marion Reid, New York Blood Center b Flow cytometry: two peaks

0 0