Exeley Inc.

Khan, Zeba

Ali, Sharique A

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A preliminary study assessing the effect  
of isocyanate in neuroblastoma brain cells in vitro
Zeba Khan and Sharique A Ali*
Department of Biotechnology, Saifia Science College, Bhopal, India,  
* Email: drshariqueali@yahoo.co.in
Isocyanate is an intermediate compound used in the manufacturing of a number of pesticides. The aim of this study is to understand 
the mechanism of isocyanate in SHSY‑5Y neuroblastoma cells in vitro. Cells were treated with a chemical equivalent of isocyanate, 
i.e., N‑succinimidyl N‑methylcarbamate (NSNM). Cell cytotoxicity, as well as qualitative and quantitative alpha‑synuclein protein 
levels were analyzed using different molecular techniques. NSNM at a concentration of 0.005 µM significantly increased cell death, 
in a time‑dependent manner, as well as levels of alpha‑synuclein protein in SH‑SY5Y cells. These findings demonstrate the ability 
of low doses of isocyanate to increase neuronal vulnerability by inducing cell cytotoxicity and protein dysfunction in vitro. 
Key words: pesticides, isocyanate, neurotoxic, N‑succinimidyl N‑methylcarbamate, exposure
INTRODUCTION
In today’s world, understanding the effects on hu‑
man health of occupational exposure to hazardous 
agents is a primary concern. A number of diseases such 
as cancer, diabetes, neurological impairment and lung 
diseases are associated with such exposures (Gangemi 
et al., 2016). There is a lack of human health risk as‑
sessments regarding exposure to natural and environ‑
mental hazards. A very limited number of studies have 
shown that isocyanates and their derivatives may have 
injurious health effects, while the molecular mecha‑
nisms behind such effects have yet to be addressed.
Isocyanate is an intermediate chemical used in 
the production of carbamate pesticides and includes 
methomyl, carbaryl, carbofuran and aldicarb. It is 
widely used in the formation of polyurethane forms, 
paints, insecticides, varnishes and elastomer industries 
(Hon et al., 2017). Methyl isocyanate has been found in 
the smoke from several types of cigarettes at 1–5 ppm. 
Screening for adverse effects of pesticides and their 
intermediates is very important from a health per‑
spective, as these pesticides are used on a large scale 
and the primary mode of pesticide application is gen‑
erally through aerosols, which can be inhaled easily 
and readily absorb into soils. To the best of our knowl‑
edge, no studies have been done to assess the effect of 
isocyanate on brain cells. In view of this, the aim of 
this study is to understand the molecular mechanisms 
induced by isocyanate using neuroblastoma cells in vi-
tro. Isocyanate is highly toxic and not readily available 
in markets, thus N‑succinimidyl N‑methylcarbamate 
(NSNM; containing a functional isocyanate group) was 
used in this study to understand the mode of action 
of this compound in cultured SH‑SY5Y neuroblastoma 
cells in vitro.
METHODS
Dulbecco’s modified Eagle’s medium (DMEM)/
Nutrient F‑12Ham, fetal bovine serum (FBS), antibi‑
otic‑antimycotic solution, MTT [(3‑(4,5‑dimethylth‑
iazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide)], and 
NSNM were procured from Sigma Aldrich, and al‑
pha‑synuclein primary antibody and horseradish per‑
Received 24 September 2020, accepted 29 March 2021
RESEARCH PAPER
Acta Neurobiol Exp 2021, 81
DOI: 10.21307/ane‑2021‑018
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provided by Exeley Inc.
Khan and Ali Acta Neurobiol Exp 2021, 81
oxidase (HRP) conjugated secondary antibody from 
Santa Cruz Biotechnology. 
Cell culture grade chemicals were procured from 
Himedia. Culture flasks, 96 ELISA plates and petri dish‑
es were procured from BD falcon.
Cell culture condition and NSNM exposure
SH‑SY5Y cells were gifted by Dr. Amit Deshpandey 
and were grown in DMEM/F‑12Ham media with 10% FBS 
and 1% antibiotic/antimycotic solution in a humidified 
environment containing 5% CO2 and 95% air at 37°C as 
per ATCC catalogue instructions. The study was designed 
in a time‑dependent manner. Dimethyl sulfoxide (DMSO) 
was used to prepare a 1 M stock solution of NSNM. At 
75% cell confluency, cells were treated with a 0.005 µM 
concentration of NSNM for 24, 48 and 72 h. For each pa‑
rameter, untreated cells were used as a control.
Assessment of cytotoxicity
5×103 cells were seeded onto 24‑well plates and kept 
overnight at 37°C in a 5% CO2 incubator.
The next day, cultured cells were treated with 
0.005 µM NSNM and incubated for 24 h, 48 h and 72 h. 
After the appropriate exposure time, cells were harvest‑
ed and washed with phosphate‑buffered saline (PBS). An 
equal volume of trypan blue solution (0.4% in PBS) and 
cell suspension were mixed and incubated for 10 min. 
After incubation, cells were loaded on a Neubauer cham‑
ber and counted under a microscope. Cell death was cal‑
culated as the percentage of trypan blue positive cells in 
the total population of stained and unstained cells.
Cell cytotoxicity can be determined using yellow MTT 
dye. Living cells reduce the MTT to purple formazan with 
the help of mitochondrial dehydrogenase enzymes. Cells 
were plated on 96‑well culture plates at a density of 5 × 103 
cells/well. After overnight incubation, cells were treat‑
ed with 0.005 µM NSNM. After the exposure time, cells 
were then incubated with MTT (0.5mg/mL) for 3–4 h. The 
formazan particles were then solubilized with 200 µl di‑
methyl sulfoxide and kept on an orbital shaker for 5 min. 
The absorbance was recorded at 570 nm through an ELI‑
SA reader. The results were expressed as viable cell per‑
centage with respect to an untreated control.
Western blot for the detection of alpha‑synuclein 
protein in SH‑SY5Y cells
Western blot assay was performed to examine the al‑
pha‑synuclein protein expression level in SH‑Y5Y cells. 
Cells were seeded and treated with 0.005 µM NSNM. Af‑
ter different time points of incubation, cells were washed 
with PBS. Lysis was carried out using ice cold cell lysis 
buffer containing 1% triton X‑100 (prepared in 0.1 M so‑
dium phosphate buffer), 0.1 mM phenylmethylsulfonyl 
fluoride (PMSF) or 100 µl of protease inhibitor cocktail 
at 4°C for 30 min. The cell lysates were centrifuged at 
1,000 × g for 10 min at 4°C. Concentration of protein was 
determined by the Bradford method. After determining 
the concentration of protein, extracted protein was load‑
ed on to a 10% gel for SDS‑PAGE. Blots were hybridized 
with antibodies against glyceraldehyde‑3‑phosphate 
dehydrogenase (GAPDH; dilution 1:1000) and against al‑
pha‑synuclein (1:2000) overnight at 4°C. The membranes 
were then washed and further hybridized with secondary 
antibody tagged with HRP (1:3000) for 1 h and washed 
twice with phosphate buffer saline‑Tween 20 (PBST). The 
blots were developed using a suitable substrate and the 
expression of protein was visualized.
Indirect immunofluorescence for the detection  
of alpha‑synuclein protein in SH‑SY5Y cells
Through an indirect immunofluorescence assay, ex‑
pression of alpha‑synuclein protein was determined in 
SH‑SY5Y cells. 2 × 104 cells were grown on glass cov‑
er slips for 24 h. After incubation, media was changed 
and 0.005 µM NSNM was added for 24, 48 and 72 h. Af‑
ter incubation, cells were washed with PBS, followed 
by fixation with 3% paraformaldehyde for 20 min and 
then permeabilized with 0.1% triton X‑100 for 10 min. 
Cells were blocked with 10% BSA for 30 min. Primary 
antibody at a dilution of 1:1000 was added and the cells 
were incubated for 1 h. Cells were washed with 0.1% 
PBST. After washing, cells were incubated with fluo‑
rescein isothiocyanate (FITC) conjugated secondary 
antibody (dilution 1:40) for 2 h, preferentially in dark. 
Again, cells were washed with 0.1% PBST. The cover slip 
was mounted and viewed, followed by photographing 
using an inverted fluorescence microscope.
Quantification of alpha‑synuclein in SH‑SY5Y cells 
by cell based ELISA
5 × 103 cells/well were incubated in 96‑well plates. 
After overnight incubation, cells were treated with 
NSNM for different time points. After the exposure 
time, cells were fixed with 0.1% formaldehyde in PBS. 
After removing the formaldehyde, the wells were gen‑
tly washed with PBS three times. Methanol was add‑
ed for 5 min. Blocking was carried out with 3% serum. 
Wells were washed with PBS. Alpha‑synuclein primary 
Effect of pesticide on cultured brain cellsActa Neurobiol Exp 2021, 81
antibody was added (1:1000) for 2 h. Wells were washed 
with PBST 0.01% and further incubated with second‑
ary antibody (1:5000) for 30 min. The cells were washed 
with PBST, as earlier, and TMB (3,3ʼ,5,5ʼ‑tetramethyl‑
benzidine) was added to each well. 2N H2SO4 was used 
to stop the reaction and absorbance at 440–460 nm was 
taken using a plate reader (BMG Fluostar).
Statistical analysis
All experiments were done in duplicate. Statistical 
differences were analyzed using Student’s t‑test and 
a p‑value ≤0.05 was considered statistically significant.
RESULTS
NSNM’s effects on the viability of SH‑SY5Y cells
When SHSY‑5Y cells were treated with 0.005 µM 
NSNM, significant time‑dependent effect on cell death 
was observed as compared to control (p<0.05) and mea‑
sured by trypan blue exclusion assay (Fig. 1A). In addi‑
tion, MTT assay results revealed that from 24 h to 72 h 
cell viability was significantly decreases as compared 
to control (p<0.05; Fig. 1B).
Qualitative analysis of alpha‑synuclein protein 
expression in SH‑SY5Y cells treated with NSNM
When SH‑SY5Y cells were exposed to 0.005 µM 
NSNM for different time points, increased band inten‑
sity was observed with increasing incubation time as 
compared to control using the western immunoblot‑
ting technique (Fig. 2). GAPDH was used as a loading 
control. Indirect immunofluorescence results revealed 
elevated expression of alpha‑synuclein protein under 
40x magnification as compared to control. At 48 and 
72 h the cell morphology of SHSY‑5Y cells had also 
changed as compared to control (Fig. 3).
Qualitative analysis of alpha‑synuclein protein  
in SH‑SY5Y cells after treatment with NSNM
To quantify the expression of alpha‑synuclein pro‑
tein, cell‑based ELISA was performed and the results 
showed that, with an increasing incubation time for 
cells treated with 0.005 µM NSNM, the level of alpha‑sy‑
nuclein significantly increased as compared to control 
(p<0.05; Fig. 4).
DISCUSSION
Studies have reported that pesticides are associated 
with human health and environmental concerns. 
Effects that have been linked with pesticides and 
herbicides in humans include skin, brain, lungs and 
A B
Fig. 1. (A) Cell death through trypan blue assay. Cell death significantly increased with increasing incubation time. *p<0.05. (B) Cell death determined by 
MTT assay. Cell death significantly increased with increasing incubation time. *p<0.05.
Fig. 2. Western blot analysis showing increased band intensity of alpha‑sy-
nuclein protein with increasing incubation time.
Khan and Ali Acta Neurobiol Exp 2021, 81
stomach changes. Additionally, occupational or acci‑
dental exposures to pesticides can lead to urgent hospi‑
talization and even death. Screening of such pesticides 
and understanding the underlying mechanisms caus‑
ing illness is very important for intervention.
Isocyanate is an intermediate chemical used in the 
production of a number of pesticides. To our knowl‑
edge, isocyanate has not previously been studied to 
determine its effect in brain cells. In the present study 
we used cultured SH‑SY5Y neuroblastoma cells to un‑
derstand the mode of action of isocyanate. We used 
a chemical equivalent of isocyanate (NSNM) to study 
the effect in SH‑SY5Y cells. We screened for cytotox‑
icity of NSNM as wells as levels of alpha‑synuclein in 
cultured brain cells after exposure for different time 
periods. The cellular toxicity of NSNM on SH‑SY5Y 
cells was assessed by the trypan blue and MTT assays. 
Our data showed that NSNM at the concentration of 
0.005 µM induced cytotoxicity in a time‑dependent 
manner. Previous studies reported that NSNM in‑
duces cell death in mouse‑derived cell lines, as well 
as in yeast, and revealed that the apoptotic pathway 
and DNA damage proteins were involved in cell death 
mechanisms (Mishra et al., 2008). In addition, Azad et 
al. (2014) conducted an experiment on budding yeast 
and identified NSNM as an epigenetic modifier that 
forms an adduct with histone and leads to decreased 
histone acetylation. 
Methyl isocyanate gas, which is known for the 
world’s worst exposure disaster in Bhopal, India, 
also caused genotoxicity in rat brain cells in vivo in 
a dose‑dependent manner (Anderson et al., 1990). 
A case‑control study conducted by Chuang et al. 
(2017) revealed that people exposed to carbamate pes‑
ticides were at a higher risk of developing Parkinson’s 
disease.
In our study, immunofluorescence results demon‑
strated a change in the morphology of SHSY‑5Y cells 
with increasing incubation time with NSNM. Our find‑
ings are in agreement with the finding of Chang and 
colleagues (2006), which found that 50 µM of aldicarb 
and carbaryl significantly decreased neurite length in 
neuroblastoma cells as compared to control. Our MTT 
assay results revealed cell cytotoxicity upon exposure 
to NSNM. Ruiz et al. (2006) conducted an experiment 
on Chinese hamster ovary cell lines and showed that 
mitochondrial integrity was disturbed upon exposure 
to seven different carbamate pesticides using the 
MTT assay.
In the present study, expression of alpha‑synuclein 
protein was also studied in SH‑SY5Y cells after expo‑
sure to NSNM. Many neurodegenerative diseases are 
characterized by the abnormal accumulation of ag‑
gregated proteins in the brain. Thus, we analyzed the 
expression of alpha‑synuclein protein quantitatively, 
as well as qualitatively, by Western blot, indirect im‑
munofluorescence and cell‑based ELISA, respectively. 
Our results showed that with increasing incubation 
time the expression of alpha‑synuclein protein also 
increased. Several studies (Viller‑Pique et al., 2016; 
Xicoy et al., 2017) have been documented describing 
that certain pesticides increase the level of alpha‑sy‑
nuclein protein in SH‑SY5Y cells. In this study, it was 
Fig. 4. Cell‑based ELISA assays showed that with increasing incubation times 
of SH‑SY5Y cells with NSNM, the levels of alpha‑synuclein expression were 
also increased significantly as compared to control. *p<0.05.
Fig. 3. Indirect immunofluorescence images showed elevated levels of alpha‑synuclein protein from 24 h to 72 h as compared to control.
Effect of pesticide on cultured brain cellsActa Neurobiol Exp 2021, 81
noted that with increasing incubation time, increased 
cell death, as well as increased levels of alpha‑synucle‑
in protein, were observed. Several studies have report‑
ed that aggregation of alpha‑synuclein makes neurons 
more prone to cell death (Yasuda et al., 2013; Vila and 
Przedborski, 2003). Our results are in agreement with 
other studies where cells treated with different class‑
es of pesticides demonstrated an overexpression of 
alpha‑synuclein, which led to mitochondrial dysfunc‑
tion and cell death in various cultured cell lines (Nor‑
ris et al., 2007; Chorfa et al., 2013). A study conducted 
by Ramalingam et al. (2019) demonstrated that com‑
plex and disrupted alpha‑synuclein protein dynamics 
were involved in neurodegeneration in response to the 
pesticide rotenone through western blot analysis of al‑
pha‑synuclein protein. Another study explained that 
rotenone induced an upregulation of alpha‑synuclein 
protein levels through the stimulation of their de novo 
synthesis rather than through a reduction of their 
chaperone‑mediated autophagy degradation (Sala et 
al., 2013).
CONCLUSION
These findings represent the ability of low doses of 
isocyanate to increase the vulnerability of neurons by 
inducing cell cytotoxicity and protein dysfunction. The 
screening of environmental contaminants capable of 
causing deregulation of neuronal function and/or trig‑
ger brain‑related disorders would be of significant ben‑
efit in controlling exposures and likely reducing the 
incidence of neurological impairments. Furthermore, 
additional studies are required to understand the inter‑
nal signaling pathways associated with neural damage 
from exposure to isocyanate.
ACKNOWLEDGEMENT
We sincerely acknowledge Dr Amit Deshpandey for 
providing lab facility for my work.
REFERENCES
Anderson D, Goyle S, Phillips BJ, Tee A, Beech  L, Butler WH (1990) Ef-
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Azad GK, Singh V, Tomar RS (2014) Assessment of the biological pathways 
targeted by isocyanate using N‑succinimidyl N‑methylcarbamate in bud-
ding yeast Saccharomyces cerevisiae. PLoS One 9: e92993.
Chang PA, Wu YJ, Li W, Leng XF (2006) Effect of carbamate esters on neu-
rite outgrowth in differentiating human SK‑N‑SH neuroblastoma cells. 
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Chorfa A, Bétemps D, Morignat E, et al. (2013) Specific pesticide‑dependent 
increases in α‑synuclein levels in human neuroblastoma (SH‑SY5Y) and 
melanoma (SK‑MEL‑2) cell lines. Toxicol Sci 133: 289–297
Chuang CS, Su HL, Lin CL, Kao CH (2017) Risk of Parkinson disease after 
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Hon CY, Peters CE, Jardine KJ, Arrandale VH (2017) Historical occupational 
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A preliminary study assessing the effect of isocyanate in neuroblastoma brain cells in vitro

Gupta, A.

Chaudhary, K.

Asati, S.

Kakkar, B.

IMMUNOHEMATOLOGY, Volume 37, Number 2, 2021 69
The Lewis blood group system is unique because antigens are 
neither alleles of the same gene nor are they synthesized by 
red blood cells (RBCs); rather, they are adsorbed onto the RBC 
membrane from plasma as glycolipids. Antibodies against Lewis 
antigens are predominantly naturally occurring immunoglobulin 
(Ig)M type that sometimes react at 37°C and the antihuman 
globulin phase. Lewis compound antigens, ALeb and BLeb, have 
been described that were confirmed because of the presence 
of antibodies against them. These compound antigens are the 
result of an interaction between ABO, H, SE, and LE genes. 
Immunohematology 2021;37:69–71. DOI: 10.21307/
immunohematology-2021-010.
Key Words: Lewis blood group system, compound antigen, 
ALeb, immunohematology
The Lewis blood group system (Le) is unique because it 
is the only system in which the antigens are not synthesized 
by red blood cells (RBCs); rather, the antigens are passively 
adsorbed onto the RBC membrane.1 Le antigens are soluble 
carbohydrate moieties formed by tissue cells and secreted 
by body secretions like saliva, where they appear as 
glycoproteins; in plasma, however, they appear as glycolipids. 
The Le phenotype depends on ABH secretor status of an 
individual, although FUT2 and FUT3, the secretor (SE) gene 
and LE gene on locus 19q and 19p, respectively, are inherited 
independently.2 Genetic interaction also exists between the LE 
and ABO genes because the amount of Le antigen detectable 
on the RBC is influenced by the ABO genes inherited, and the 
products of ABO and LE share the same precursor substrate. 
Individuals who are Le(a+b–) are nonsecretors, Le(a–b+) are 
secretors, and Le(a–b–) can be secretors or nonsecretors. 
Although individuals who are Le(a+b–) are nonsecretors, 
Lea substance is still secreted regardless of secretor status. 
An individual can be a nonsecretor (sese) of ABH and still 
secrete Lea into body fluids, producing the phenotype Le(a+) 
on the RBCs. Frequency of the Le phenotypes in the Indian 
population are as follows: Le(a+b–) 20.82 percent, Le(a–b+) 
60.57 percent, and Le(a–b–) 18.61 percent.3 Compound 
antigens in the Lewis blood group system include ALeb and 
BLeb, which are the result of interaction between ABO, H, SE, 
and LE genes.4 Antibody against these compound antigens 
(e.g., anti-ALeb) has been shown to react only when RBCs 
possess both A and Leb together.1
Here, we describe the immunohematology workup of a 
rare and interesting case of incompatible crossmatches with 
negative indirect antiglobulin test and the antibody identified 
as anti-A1Leb.
Case Report
A 61-year-old male patient was admitted to a local 
hospital with complaints of hematemesis, abdominal 
distension, decreased appetite, weight loss, weakness, and 
itchy and yellow discoloration of the skin. He was a known 
chronic alcoholic and was diagnosed with hepatitis C in 2010. 
Baseline investigations at the time of admission revealed low 
hemoglobin (Hgb) of 5 g/dL (normal range 13.5–17.5 g/dL 
for men), deranged liver function tests with markedly raised 
bilirubin and enzymes, abnormal coagulation profile with 
raised international normalized ratio, low total protein, low 
serum albumin, and negative α-fetoprotein. Because of the 
low Hgb (5 g/dL), a request was sent for packed red blood 
cell (PRBC) transfusion. The hospital blood bank performed 
routine antibody screening and reported a positive result with 
incompatible PRBC crossmatch. Because of limited resources, 
the patient’s samples were referred to the Department of 
Transfusion Medicine in our hospital for further antibody 
identification workup, and 2 units of antigen-negative 
antihuman globulin (AHG) crossmatch-compatible PRBCs 
were requested. Before his referral, the patient had been 
transfused with 1 unit of PRBCs 3 years prior and with 1 unit 
2 weeks prior.
The patient’s RBCs were typed as group A, D+ with a 
positive autocontrol (2+; AHG phase). Direct antiglobulin tests 
(DATs) with polyspecific and monospecific AHG reagents 
were positive (poly = 2+; immunoglobulin [Ig]G = 2+; C3d = 
2+). Indirect antiglobulin test (IAT) with a three-cell screening 
panel was negative. Two randomly selected PRBC units 
crossmatched by column-agglutination technology (CAT) 
were found to be incompatible (2+). A repeat crossmatch was 
performed using a fresh sample with group A, D+; group O, 
D+; and group A2, D+ PRBC donor units. Tables 1–6 briefly 
Anti-A1Le
b: a mind boggler
A. Gupta, K. Chaudhary, S. Asati, and B. Kakkar
Case RepoRt
70 IMMUNOHEMATOLOGY, Volume 37, Number 2, 2021
A. Gupta et al.
summarize the immunohematology workup performed. 
(Reagent RBC panel, antisera for phenotyping, CAT gel cards, 
DAT cards, DTT, enzymes, and 22% bovine albumin were 
from Bio-Rad, Hercules, CA; Anti-A1 from J. Mitra, New 
Delhi, India.) After detailed workup, a suspicion of a compound 
antibody directed against antigens in two blood group systems 
(i.e., ABO and Lewis blood group systems) was raised. The 
offending antibody was identified as anti-A1Leb, and 2 units 
of group A1, Le(b–) AHG crossmatch-compatible PRBCs were 
found and issued to the patient (Table 6). No adverse events 
were reported during or after transfusion.
Discussion
Le antigens are not alleles of the same gene. Their synthe-
sis depends on the interaction of the enzymes (transferases) 
produced by the LE and SE genes. The Lewis and secretor 
transferases fucosylate (add fucose) Type 1 chains, which 
are predominant types in secretions and plasma and refer to 
the beta linkage of the first carbon of galactose to the third 
carbon of N-acetylglucosamine (13 linkage) residue of 
the precursor structure.4 The SE gene codes for the enzyme 
(α1,2-L-fucosyltransferase), which adds terminal fucose to 
the Type 1 chain to form Type 1H. The Le allele codes for 
α1,4-L-fucosyltransferase, which transfers L-fucose to the 
Type 1H chain on glycoprotein or glycolipid structures to 
form Leb. Small amounts of Lea are made before the secretor 
enzyme can add the terminal fucose. If these individuals also 
have A or B genes, Type 1H structures will be converted to A or 
B structures and the Le fucosyltransferase will then produce 
ALeb or BLeb.1,2
Le(a–b–) phenotype is not the result of the absence of 
Lewis antigens on the RBCs caused by the absence of the 
Lewis gene (FUT3), but rather by explicit point mutations 
in the FUT3 and other genes.5,6 Although serologically 
Table 1. Basic immunohematologic workup 
Testing Result
Blood group/D type A1, D+
DAT IgG: 2+, C3d: 2+
IAT Negative
Autocontrol (AHG)* 2+
Anti-A1 lectin Positive (4+)
DAT eluate† against A, D+ RBCs 3+
DAT eluate† against O, D+ RBCs Negative
DAT eluate† against O, D– RBCs Negative
*Cell separation using microhematocrit tubes.
†Heat elution.
DAT = direct antiglobulin test; Ig = immunoglobulin; IAT = indirect 
antiglobulin test; AHG = antihuman globulin; RBCs = red blood cells.
Table 6. Crossmatch with phenotype-matched PRBC units 
ABO group Lewis type CAT crossmatch CTT crossmatch
A1 Le(a–b–) Compatible Compatible
A1 Le(a–b+) Incompatible (2+) Incompatible (1+)
A2 Le(a–b+) Compatible Compatible
O Le(a–b+) Compatible Compatible
PRBC = packed red blood cell; CAT = column-agglutination technology;  
CTT = conventional tube test.
Table 2. Crossmatch performed with random PRBC units
Blood group/D type of 
PRBC unit
Number of 
units tested
CAT  
crossmatch
Conventional tube crossmatch
RT 37°C
A1, D+ 6 2+ 1+ 1+
O, D+ 6 Compatible Compatible Compatible
A2, D+ 2 Compatible Compatible Compatible
PRBC = packed red blood cell; CAT = column-agglutination technology;  
RT = room temperature.
Table 3. Extended phenotype (testing on pre-transfusion sample)
D C E c e K Jka Jkb Fya Fyb M N S s Lea Leb Control*
+ + Neg Neg + Neg + Neg + + + Neg Neg + Neg Neg Neg 
*22% bovine albumin. Neg = negative.
Table 4. Inhibition studies performed using saliva
Patient serum antibody Saliva
Reactivity with A1,  
Le(a–b+) RBCs
Anti-A1Leb None Present
Anti-A1Leb A1, Le(a+b–) Present
Anti-A1Leb O, Le(a–b+) Present
Anti-A1Leb A1, Le(a–b+) Inhibited
Anti-A1Leb A2, Le(a–b+) Present
RBCs = red blood cells.
Table 5. Testing with 0.1 M DTT-treated serum
Reciprocal dilution
Test sample 2 4 8 16 32 Interpretation
Serum + DTT 2+ 1+ 0 0 0
IgM + IgG
Serum + PBS 2+ 1+ 1+ 0 0
DTT = dithiothreitol; PBS = phosphate-buffered saline; Ig = immunoglobulin.
IMMUNOHEMATOLOGY, Volume 37, Number 2, 2021 71
Compound Lewis antibody
Lewis antigens are absent on the RBCs, Lewis and ABO 
substances can be detected in the tissues and secretions of 
the Le(a–b–) secretors, depending on the secretory status and 
ABO genotype of the person. The reported prevalence of the 
Le(a–b–) phenotype in the Indian population is 18 percent.3
Our patient had a compound antibody, anti-A1Leb, 
reacting only with RBCs possessing both A1 and Leb antigens 
(i.e., against compound antigen A1Leb). Anti-A1Leb was first 
identified by Seaman et al.7 in 1968 in a patient with blood 
group A1B and the Le(a–b–) phenotype. They reported that 
the antibody reacts with A1 Le(b+) RBCs but not with RBCs 
having A1 or Leb alone.7 This antibody of compound speci-
ficity was called “Siedler.” A literature search on antibodies 
that react with RBCs containing antigens against two separate 
blood group systems includes “Magard,” which reacts against 
RBCs with secretor phenotype A1 Le(a–b–).7 Tilley et al.8 in 
1975 reported that A, B, and ALeb substances detected in the 
lipid fractions are glycosphingolipids that react with anti-A, 
anti-B, or anti-ALeb, respectively. 
Our patient’s sample tested as group A1, D+ with 
incompatible crossmatch and negative IAT. Heat elution 
of DAT+ RBCs reveals an eluate that was reactive with A1 
RBCs (2+; AHG) but was nonreactive against group O RBCs 
(Table 1). Multiple blood units were crossmatched using CAT 
and conventional tube testing (Table 2); among them, all group 
A1, D+ RBC units were found to be incompatible, whereas all 
group O, D+ and A2, D+ blood units were compatible with 
patient’s serum. Ruling out all donor- and patient-related 
causes of incompatible crossmatch (both at room temperature 
and 37°C) with a negative IAT, the diagnosis was narrowed 
down to a compound antibody reacting against two different 
antigens. Such a compound antibody was identified as anti-
A1Leb, which reacts only in the presence of both A1 and Leb 
antigens (Table 6).
Lewis antibodies are generally naturally occurring IgM 
antibodies that sometimes react at 37°C and AHG phase, 
although weakly, and are enhanced by enzyme treatment. Our 
patient has mixed IgM + IgG antibody (Table 5), which was 
clinically significant. Patient Lewis phenotype, performed by 
cell separation using microhematocrit tubes, was Le(a–b–), as 
seen in Table 3; a person with this phenotype could produce 
anti-Leb. Table 4 shows that reactivity of anti-A1Leb with 
group A1, Le(a–b+) RBCs can be inhibited with group A1, 
Le(a–b+) saliva but not with group O Le(a–b+), A2 Le(a–b+), 
A1 Le(a–b–), or A1 Le(a+b–) saliva. One group A1, Le(a+b–) 
PRBC unit was typed, crossmatched, and transfused to the 
patient; the post-transfusion period was uneventful.
We find this case interesting, since the immunohematol-
ogy workup created confusion about the type and nature of 
the antibody. This case report also highlights the importance 
of using the traditional crossmatch at the AHG phase because 
reactivity could be missed with the electronic crossmatch or 
type-and-screen protocol.
Acknowledgments
We are thankful to our senior residents, Drs. Varuni 
Yadav, Anupa Pokhrel, Prerna Sachdeva, and Harkiran Arora, 
for reviewing this manuscript.
References
 1. Leger RM, Novotny AK. Blood group terminology and 
common blood groups: the Lewis, P, I, MNS, Kell, Duffy, Kidd 
and Lutheran. In: Harmening DM, Ed. Modern blood banking 
& transfusion practices. 17th ed. Philadelphia, PA: F.A. Davis, 
2019:173–211.
 2. Olsson ML, Westman JS. ABO and other carbohydrate blood 
group systems. In: Cohn CS, Delaney M, Johnson ST, Katz LM, 
Eds. AABB technical manual. 20th ed. Bethesda, MD: AABB, 
2020:313–5.
 3. Thakral B, Saluja K, Sharma RR, Marwaha N. Phenotype 
frequencies of blood group systems (Rh, Kell, Kidd, Duffy, 
MNS, P, Lewis, and Lutheran) in north Indian blood donors. 
Transfus Apher Sci 2010;4:17–22.
 4. Henry S, Oriol R, Samuelsson BO. Lewis histo-blood group 
system and associated secretory phenotypes. Vox Sang 
1995;69:166–82.
 5. Henry SM. Review: phenotyping for Lewis and secretor histo-
blood group antigens. Immunohematology 1996;12:51-61.
 6. Elmgren A, Börjeson C, Svensson L, Rydberg L, Larson G. 
DNA sequencing and screening for point mutations in the 
human Lewis (FUT3) gene enables molecular genotyping of 
the human Lewis blood group system. Vox Sang 1996;70:97.
 7. Seaman MJ, Chalmers DG, Franks D. Siedler: an antibody 
which reacts with A1Le (a–b+) red cells. Vox Sang 1968;15: 
25–30.
 8. Tilley CA, Crookston MC, Brown BL, Wherrett JR. A and B 
and A1Le
b substances in glycosphingolipid fractions of human 
serum 1. Vox Sang 1975;28:25–33.
Anubhav Gupta, MD (corresponding author), MD Transfusion 
Medicine, Assistant Professor, Department of Transfusion Medicine, 
All India Institute of Medical Sciences, Jodhpur, Rajasthan, India 
342005, dr.anubhavgupta@yahoo.com; Kiran Chaudhary, MCH, 
Department Head, Department of Transfusion Medicine, Atal Bihari 
Vajpayee Institute of Medical Sciences and Dr. Ram Manohar Lohia 
Hospital, New Delhi, India; Swati Asati, DNB, Atal Bihari Vajpayee 
Institute of Medical Sciences and Dr. Ram Manohar Lohia Hospital, 
New Delhi, India; and Brinda Kakkar, DNB, Assistant Professor, 
Transfusion Medicine, Bharati Vidyapeeth University, Pune, 
Maharashtra, India.


Anti-A1Leb: a mind boggler

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Effect of cryopreservation on a rare McLeod donor red blood cell concentrate

Dreyer, Craig

Australasian Orthodontic Journal Volume 37 No. 1 May 2021 1
Editorial
Standing still means falling behind
In 2017, the Australasian Orthodontic Journal 
celebrated its 50th anniversary. Since its inception, 
its constitutional charter has been to publish twice 
per annum and provide news, information and 
record events promoted by the Australian Society of 
Orthodontists. As the mouthpiece of the Society, this 
brief has largely remained in-house, self-contained 
and unchanged.
A retired academic from Manchester, in a weekly 
blog,1 often laments that the orthodontic literature 
is shielded from general access by subscription and 
member protection. It is firmly believed that all 
information should be freely available so that the 
orthodontic world and the treated public benefit. The 
difficulty of literature access creates inconvenience 
for researchers, academics and clinicians who are 
unable to retrieve relevant publications and contact 
colleagues on matters of mutual interest. Unless 
libraries subscribe to journals, readers are unable 
to obtain specific articles without the payment of 
substantial fees.
Like most orthodontic journals, the AOJ has remained 
closed from general access and only subscribers receive 
the latest clinical and research information. The 
notable journal exception is the Angle Orthodontist, 
which changed to an open access format many years 
ago and remains the only major orthodontic journal 
that has a non-commercial, non-profit publisher. 
Its Editorial Board states that the ability to operate 
exclusively in the best interests of readers and authors 
is valued and sponsored. The journal’s website is 
completely free and open to all visitors. Not so for 
other mainstream orthodontic journals.
While the AOJ has not changed its publishing 
philosophy, the publishing industry has advanced 
significantly following the advent of computerisation. 
On-line access to news and information, the explosive 
increase in the use of social media and improved file 
transfer systems have fundamentally changed the way 
that the publishing world operates. Journal boards 
have basic obligations to four entities, namely the 
owner of the journal (ASO), the submitting authors, 
the readership (subscribers) and other associated 
bodies (advertisers, lawyers, public).
Currently, submitting authors are severely handi-
capped by the AOJ’s publishing timetable and many 
have indicated that further submissions will not be 
considered. Researcher academic advancement is not 
promoted by a delayed publication of papers. This 
could be managed by a more frequent (quarterly) 
publishing timetable but this would double current 
publishing costs and, since the journal is a non-profit 
entity, would not be viable.
Promoted by the Journal’s Board, the intention is to 
take the AOJ into the open access world so that all 
involved parties are treated as fairly as possible. The 
benefits of open access to the literature are many-fold 
and include greater access and author ability to address 
a wider audience. The reach of the articles or materials 
increases significantly since readers more easily 
retrieve papers, therefore greater exposure is achieved. 
In addition, research results can be made immediately 
available within the scientific community. Authors do 
not have to wait an inordinately long time for their 
work to appear.
The greater access to knowledge spurs interdisciplinary 
and multidisciplinary research collaboration and open 
access articles tend to have a much greater impact 
in the short-term compared with ‘subscription-only’ 
work. Furthermore, additional studies show a slightly 
larger long-term impact for open access articles. An 
important aspect is that listing will be facilitated on 
PubMed, which will attract authors who have deserted 
the AOJ over recent years.
Members will find an article more easily located and 
available in the open access domain, rather than 
through the current citation management system. 
More readers become aware of authors who publish 
in open access journals as opposed to subscription-
only journals. Institutions enhance their profile by 
Australasian Orthodontic Journal Volume 37 No. 1 May 20212
participating in or hosting open access publishing. 
Funding agencies supporting the research achieve 
more prominence.
Open access publications are usually less expensive 
to produce and disseminate, therefore journals and 
publishers benefit. It is well known that open access 
increases visibility and attracts subscriptions.
The AOJ has stood still and, of the many global 
orthodontic publications, the journal barely receives 
a mention when impact comparisons are made. The 
intention is to establish an open access format so that 
the profile of the journal is enhanced. This then is the 
last hard copy issue and all of the contained papers will 
be available on-line in one main volume to be added 
to as papers are submitted, reviewed and prepared for 
immediate uploading. 
Open journal access is a much-needed change and 
great opportunity to ensure that the AOJ receives the 
global exposure and recognition it rightly deserves.
Craig Dreyer
1 https://kevinobrienorthoblog.com


Standing still means falling behind

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