hemoglobin oxygen desaturation in the absence of acute illness is common in children with sickle cell anemia ( sca ) , and is associated with higher cerebral blood flow velocities [ 1 , 2 ] , and with risk of complications including stroke .
the underlying mechanisms of hemoglobin oxygen desaturation in sca are poorly understood but may involve the severity of anemia   as well as differences in hemoglobin oxygen affinity compared to hemoglobin a ( hba ) , with increased expression of 2.3 dpg in hemoglobin s ( hbs ) resulting in a right - shifted hemoglobin oxygen affinity curve and other differences in red cell physiology .
other potential causes include a history of acute chest syndrome and reduced pulmonary   and cardiac function .
coinheritance of alpha - thalassemia deletions and glucose-6-phosphate deficiency ( g6pd ) may affect the degree of anemia [ 8 , 9 ] whilst alpha - thalassemia status modifies red cell indices [ 1012 ] and rheology , as can iron status .
we therefore investigated potential hematological , genetic , and nutritional predictors of daytime hemoglobin oxygen saturation in tanzanian pediatric patients homozygous for hbs ( sca ) and in non - sca local controls .
ethical permission was granted by the muhimbili university of health and allied sciences ethics committee ( mu / rp / aecnoi.xii/77 ) . written informed consent was obtained from parents or guardians in their own language .  
children ( less than 17 years ) with sca ( hbss genotype ) were enrolled in the scd cohort study at muhimbili national hospital , dar - es - salaam .
resting pulse oximetry data ( masimo radical , masimo corporation , usa ) and blood samples were collected at routine outpatient clinic visits between november 2007 and december 2008 .
analysis was limited to data collected at a single steady - state time point . a strict definition of steady state was employed ( temperature <
37.5c , no malaria parasitaemia , no reported pain , no blood transfusion within 90 days or hospital admission within 30 days on either side of the selected time point ) and determined to be clinically well by the attending doctor .
all cohort children are routinely prescribed folate supplementation ( 5  mg / day ) .  
non - sca children were those who presented for sickle testing between october 2004 and december 2008 but who had hbaa or hbas by hemoglobin electrophoresis .
none of the children had malaria parasitaemia or fever ( temperature > 37.4c ) and all were clinically well .
full blood counts were performed using an automated cell counter ( pentra 60 , horiba abx , kyoto , japan ) .
serum iron and total iron binding capacity were measured in serum samples stored at 80c ( architect c8000 , abbott , new york , usa ) .
lactate dehydrogenase ( ldh ) and bilirubin ( total and conjugated ) were measured in fresh serum samples ( architect c8000 , abbott , new york ) by muhimbili central pathology laboratory .  
children attending screening for sickle status were typed for hbs by alkaline hb electrophoresis ( helena , sunderland , tyne and wear , uk ) .
children enrolled in the muhimbili sickle cohort also had hemoglobin fractions , including hbf , quantified by hplc using the -thalassemia short programme on the variant analyzer ( biorad , hercules , ca , usa ) .
in addition , children enrolled in the muhimbili sickle cohort had hbss status confirmed by genotype and were genotyped for the 3.7 alpha - thalassemia deletion using a pcr - based method and agarose gel visualization as per published methods   and the 202- and 376-single nucleotide polymorphisms ( snps ) for glucose 6-phosphate dehydrogenase deficiency ( g6pd ) and hbs using multiplex sequenom .  
data were analyzed using stata 11-ic ( statacorp , college station , tx , usa ) .
daytime spo2 is not normally distributed due to an excess of observations having the maximum possible value of 100% and a skewed distribution towards the lower values .
thus we investigated the use of negative binomial regression of count data using a new variable of spo2 - 100 compared to zero - inflated negative binomial regression .
vuong tests   indicated no strong consistent preference for either models across the explanatory variables tested . as we had no prior hypotheses that mechanisms to predict 100% versus < 100% spo2 may differ from mechanisms underlying the degree of desaturation , we selected the negative binomial regression model .
we next compared the results of these models to those from simple linear regression of the nontransformed data and observed no major differences in the results . whilst analysis of the residuals from the linear regression models indicated some skew , this
was not judged to be sufficient to render the results invalid , which were in agreement with those from the better fitting negative binomial regression models .
thus for ease of presentation and interpretation the results of the linear regression models are presented .
complete hematological , genetic , and iron status ( transferrin saturation ) data were available for 458 sca children .
in addition , complete hematological and pulse oximetry data were available for 394 non - sca children , although transferrin saturation data were only available in a small subset of 62 children and genotyping was not conducted in these controls .
pulse oximetry , hematological and iron status data for the two groups of children are summarized in table 1 . of the 458 sca children , 239
were boys ( 52% ) with a mean age of 9.7  y ( sd 4.3  y ) compared to 212 boys ( 54% ) in the 394 non - sca children with a mean age 7.0  y ( sd 4.7  y ) . in the sca children median daytime spo2 was 97% ( iq range 9499% ) ( table 1 ) , significantly lower ( wilcoxon rank sum test ; p < 0.0001 ) than in the non - sca children ( median 99% ; iq range 98100% ) ( table 1 and figure 1 ) .
hemoglobin concentration , red cell indices ( red blood cell count ( rbc ) , mean cell hemoglobin ( mch ) , and mean cell hemoglobin concentration ( mchc ) , mean cell volume ( mcv ) ) , and markers of hemolysis ( ldh and unconjugated bilirubin ) were significantly different between the sca and non - sca groups ( table 1 ) .
the proportion of sca children with low transferrin saturation ( < 16% ) indicating probable iron deficiency , was 28% ( 126/458 ) but was lower than that in non - sca tanzanian control children ( 32/62 , 52% ) ( table 1 ) . only 9 ( 2% ) of the sca and none of the non - sca patients had high transferrin saturation ( > 55% ) which might indicate iron over - load .
forty - one percent of the sca children ( 192/458 ) were heterozygous for the 3.7 alpha - thalassemia deletion and 17% were homozygous ( 78/458 ) .
eleven percent of sca children were heterozygous females for both the 202 and 376 g6pd single nucleotide polymorphisms ( snps ) which results in a moderate phenotype , whilst a further 12% ( 54/458 ) of sca children were affected homozygous females or affected males .
predictors of spo2 in sca are shown in table 2 . within the sca population all of the hematological variables
higher hemoglobin , rbc , and hbf% were associated with greater spo2 , whilst higher mch , mchc , and mcv were associated with lower spo2 .
mch was the single biggest contributor to the variation in spo2 as indicated by the still modest adjusted r 
  value of 0.09 .
there was weak evidence of a negative association between transferrin saturation and spo2 . in the non - sca population
the same hematological variables were tested for associations with spo2 , as well as hb phenotype , as versus aa . however , the only predictor of spo2 in this group was a positive association for age ( beta coefficient 0.05 , p = 0.024 ) with no evidence to suggest even weak effects of any of the hematological indices .   in the sca population for whom
genotypes were available , coinheritance of the alpha - thalassemia 3.7 genotype was associated with increased spo2 , whilst co - inheritance of g6pd deficiency was associated with decreased spo2 .  
pair - wise associations between the different explanatory variables for daytime spo2 within the sca group were investigated and are presented in full in supplementary table 1  available online at http://dx.doi.org/10.1155/2013/472909 .
alpha - thalassemia genotype was highly associated with all of the hematological variables , with the number of 3.7 deletions being associated with higher hemoglobin ( pearson correlation coefficient 0.23 ) ; rbc count ( 0.55 ) ; lower mcv ( 0.24 ) , mchc ( 0.32 ) , and mch ( 0.60 ) ; and also lower hemolytic markers , particularly unconjugated bilirubin ( 0.26 ) , all with p values < 0.0001 .
interestingly , presence of the 3.7 alpha - thalassemia deletion was also associated with improved nutritional status as determined by body - mass - index z - score .
higher mch and mcv were associated with lower hemoglobin in the sca population whilst hbf% was associated with higher hemoglobin .
transferrin saturation was positively associated with hemoglobin ( 0.17 ) and also higher mch ( 0.33 ) , both p values < 0.0005 , but was not associated with mcv .
correlations between the hematological variables were similar in the non - sca population compared to sca ( data not shown ) except that higher mcv was correlated with higher hemoglobin ( 0.64 , p < 0.001 ) but was not associated with rbc count and no association was apparent between hemoglobin and mchc .
in multivariable models predicting spo2 , the inclusion of mch with alpha - thalassemia genotype resulted in a diminished and nonsignificant association for alpha - thalassemia .
however , in models including either mch or alpha - thalassemia there were an independent effect of hbf% and weak evidence of an independent effect of g6pd limited to affected males and homozygote females ( supplementary table 2 ) , but with the greatest variation explained by a model including mch and hbf% ( adjusted r 
  = 0.15 , p < 0.001 , n = 315 ) .
few studies have attempted to determine the hematological or genetic correlates of daytime spo2 . in this study
we report that higher daytime spo2 in children with sca is associated with higher red cell count and hemoglobin , but with lower mch or mchc .
coinheritance of the alpha - thalassemia 3.7 deletion in our sca population is strongly associated with increased hemoglobin , red cell count and decreased mch and mchc and also with increased spo2 , but not independently of these factors .
thus the basis of the association between alpha - thalassemia genotype and spo2 would appear to be via its effects on red cell indices . in kenyan nonsickle populations , alpha - thalassemia 3.7 deletion copy number is also associated with slightly increased hemoglobin levels and increased red cell counts , but effects on anaemia appear to be modulated by sickle trait status .
this suggests that the decreased anemia and increased red cells observed in sca plus alpha - thalassemia results from decreased rate of red cell destruction , proposed to be due to lower hbs concentration in red cells , and thus reduced hbs polymerization which is concentration dependent as well as being increased by desaturation .
this is supported by the observation that alpha thalassemia was associated with lower levels of ldh and unconjugated bilirubin as markers of hemolysis .
red cell count and hemoglobin were not associated with spo2 when adjusting for mch and thus anemia does not appear to be a direct cause of low spo2 in our population .
a beneficial effect of alpha thalassemia on daytime spo2 has also been observed in jamaican children with sca ,   but in contrast to our study , there were also independent effects of hemoglobin , whilst neither mch , or mchc was associated with spo2 .
it is interesting to note that within the non - sca population none of the tested variables were associated with spo2 .
this may have been the result of the reduced variability in spo2 in the non - sca population , or reflect sickle - specific effects of red cell indices on spo2 .
again , this contrasts with observations in older jamaican children who were hbaa ( 1518 years ) in whom hemoglobin and mcv ( but not mch or mchc ) were associated with spo2 .  
our observation of a beneficial effect of co - inheritance of alpha - thalassemia on daytime spo2 in children with sca appears to be in direct contrast to our previous observation of a negative effect on mean overnight spo2 , measured in a small group ( n = 30 ) of similar aged tanzanian children with sca .
however , this effect of alpha - thalassemia appeared to be confounded by the strong negative association between transferrin saturation and mean overnight spo2 .
we concluded that this association was most likely the result of reverse causality from nocturnal chronic and intermittent hemoglobin desaturation causing increased transferrin saturation due to upregulation of hypoxia - inducible factor , which has downstream effects of increasing iron absorption , which may also be increased by alpha - thalassemia .
thus we suggest that in a larger dataset , alpha - thalassemia may also have a beneficial effect on mean overnight spo2 .  
similar to jamaican   and american sca children , hbf% was positively and independently associated with spo2 .
it is not possible in the current study to determine if an effect of hbf% is via direct effects on increased hemoglobin oxygen affinity or through indirect effects on rates of sickling or red cell rheology .
interestingly alpha - thalassemia was also associated with increased hbf% , perhaps through increased rate of release of immature red cells from ineffective erythropoiesis .
the co - inheritance of g6pd deficiency was negatively associated with spo2 . as there was no evidence of an association between g6pd and levels of hemolytic markers , as has been observed previously in sca , the effect on spo2 is likely through another mechanism .  
this includes elevated cerebral blood flow velocities [ 1 , 2 ] and an increased risk of stroke [ 3 , 23 ] .
co - inheritance of alpha - thalassemia is protective against elevated cerebral blood flow velocities in children with sca [ 24 , 25 ] and is associated with reduced stroke risk , whilst g6pd increases the risk .
thus the mechanisms behind these associations may , at least in part , be through effects on hemoglobin oxygen saturation and subsequent endothelial dysfunction [ 27 , 28 ] as well as other potential effects of red cell deformability and endothelial adhesion .   in conclusion , in our population of tanzanian children with sca ,
the strongest predictor of lower spo2 is increased mean cell hemoglobin , which is strongly correlated with a decreased copy number of the 3.7 alpha - thalassemia deletion .
the degree of anemia , red cell count , and hemolytic markers do not independently correlate with spo2 , thus suggesting that effects of mean cell hemoglobin and the alpha - thalassemia genotype on spo2 are mediated through effects on red cell rheology .
 low hemoglobin oxygen saturation ( spo2 ) is common in sickle cell anemia ( sca ) and associated with complications including stroke , although determinants remain unknown . 
 we investigated potential hematological , genetic , and nutritional predictors of daytime spo2 in tanzanian children with sca and compared them with non - sca controls . 
 steady - state resting pulse oximetry , full blood count , transferrin saturation , and clinical chemistry were measured . 
 median daytime spo2 was 97% ( iq range 9499% ) in sca ( n = 458 ) , lower ( p < 0.0001 ) than non - sca ( median 99% , iq range 98100% ; n = 394 ) . within sca , 
 associations with spo2 were observed for hematological variables , transferrin saturation , body - mass - index z - score , hemoglobin f ( hbf% ) , genotypes , and hemolytic markers ; mean cell hemoglobin ( mch ) explained most variability ( p < 0.001 , adj r 
 2 = 0.09 ) . in non - 
 sca only age correlated with spo2 . 
 -thalassemia 3.7 deletion highly correlated with decreased mch ( pearson correlation coefficient 0.60 , p < 0.0001 ) . in multivariable models , lower spo2 correlated with higher mch ( -coefficient 0.32 , p 
 < 0.001 ) or with decreased copies of -thalassemia 3.7 deletion ( -coefficient 1.1 , p < 
 0.001 ) , and independently in both models with lower hbf% ( -coefficient 0.15 , p < 0.001 ) and glucose-6-phosphate dehydrogenase genotype ( -coefficient 1.12 , p = 0.012 ) . this study provides evidence to support the hypothesis that effects on red cell rheology are important in determining spo2 in children with sca . 
 potential mechanisms and implications are discussed . 
1. Introduction
2. Patients 
3. Results 
4. Discussion