Whole human umbilical cord blood (HUCB) is the blood that remains in the placenta and the attached umbilical cord after the delivery of a newborn. HUCB contains not only different type of stem cells but also contains other cellular and acellular substances like red blood cells (RBCs), white blood cells (WBCs), platelets and plasma [1,2,3].  Most of the occasion after the delivery of a baby placental cord blood discarded conventionally. Recently there are several potential fates of human umbilical cord blood such as i) cryopreservation of cord blood stem cells for various diseases like cancers, blood disorders, and immune deficiencies [4,5] ii) donation of cord blood for public uses such as spinal cord injury [6], iii) umbilical cord blood may use as research purpose or iv) as alternative of whole human blood [7].

Oxygen carrying capacity of whole human umbilical cord blood is different from whole human blood due to presence of fetal haemoglobin (HbF) [8]. Normally fetus live in a physiological hypoxic environment [9,10]. Studies analysing the samples of human umbilical cord blood obtain from cordocentesis and demonstrate that partial pressure pO2 and saturated oxygen SpO2 decreases 16-18th week of gestation [10-13] and reverse the oxygen tension in the advanced stage of pregnancy gestational ages ≥ 37 weeks, depending upon vascular maturity [14]. Fetal Hb contain two α and two γ subunits (HbF) while the adult haemoglobin contains two α and two β (HbA) subunits [15]. The gene express the γ chain proteins which is present at β chain locus of chromosome 11 [16]. The γ chain differs from adult β chain either by alanine or glycine at position 136 [17]. Both alanine and

glycine are neutral and non-polar amino acids which gives rise to several physiological difference in oxygen delivery in fetal circulation [18,19]. Fetal Hb has a crucial role for transporting oxygen from maternal to fetal circulation. Fetal Hb having a high oxygen affinity and low 2-3 diphosphoglycerate content (2-3 DPG) compared to HbA [20,21]. The partial pressure (P50) is 19 mm Hg, compared to 27 mm Hg for HbA, which signifies that HbF has a high affinity of oxygen [22,23]. The γ subunit of HbF has lower affinity towards 2-3 DPG because of replacement of histidine by serine in amino acid structure of γ subunits. In fetal circulation due to presence of increase HbF and low 2-3 DPG initiate higher content of haemoglobin in fetal umbilical cord blood [24]. Human umbilical cord blood (HUCB) transfusion has also shown promising results in treating the post COVID-19 anaemic patients who were suffering symptoms of anaemia after COVID-19 infection [25].

In the present study, patients of symptomatic anaemia with low haemoglobin (< 10gm/dl) those were treated by freshly collected screened human umbilical cord blood transfusion were compared with changes of Hb as per different published literature. It is also analysed mathematically in relation with to oxygen carrying capacity in respect to group of individuals. Post transfusion haemoglobin levels also monitored in study group for consecutive four months.

The aims of this study to analyse i) post-transfusion changes following transfusion of whole human umbilical cord blood in convalescence COVID-19 anaemic patients with significant anaemia in terms on effect of the Hb and oxygen carrying capacity. ii). a mathematical model on transfused cord blood versus quantity of routinely transfused adult red cells in anaemia.

This observational cross-sectional study was carried out in the Department of Regenerative Medicine, School of Tropical Medicine, Kolkata, from August 2020- December 2022 (IEC Ref. No: CREC-STM/2020-AS-27 dated on 18.08.2020). The patients who were enrolled had tested positive for COVID-19 but had been given the standard treatment for the infection.

Methodology

Procedure of Patient’s Selection: The total sample size for the study was 152 convalescent COVID-19 patients, out of which 24 patients were mild anaemic (Hb< 10gm/dl) enrolled for cord blood transfusion, 18 patients were transfused with a single unit of freshly collected screened HUCB according to availability of ABO matched cord blood for transfusion. Six patients could not be transfused as compatible units were avail to them or as they were lost to study and follow up.

Inclusion Criteria

• Convalescent COVID-19 positive patients.
• Diagnosis of anaemia.
• Age group 18 to 91 years.
• Convalescent COVID patients willing to participate in the study.

Exclusion Criteria

• Non-compliance to the study protocol and lost to follow up patients.
• Haemoglobinopathies,
• Existing other existing systemic disorder such as chronic kidney disease (CKD), diabetes mellitus (DM), chronic liver disease (CLD) etc

Procedure of Human Umbilical Cord Blood Collection, Processing, and Quality Control

HUCB was collected aseptically from screened pregnant women after obtaining donor informed consent by lower uterine caesarean section (LUCS) under regional anaesthesia [26,27,28] (Figure-1). Cord blood collection was discontinued in cases of severe prematurity or dysmaturity, if the estimated foetal weight was less than 2 kg in USG, or if the mother had a known infectious condition such as hepatitis, HIV, etc. After the birth of healthy infants, cord blood was obtained only with the knowledge and agreement of the mothers. The anaesthesiologist made sure the mother's vitals were stable before the collection began after the infant had been taken from the operating area. The doctor decided to harvest cord blood, and then the chord was quickly cleansed with a spirit/betadine solution. Blood from the umbilical cord was taken by squeezing the vein. The umbilical cord blood collection bag was fitted with a 16-G needle (Terem India 150 ml cord blood bag). For the present study a total of 22 units of human umbilical cord blood was collected from Vidyasagar State General Hospital, South 24 PGS and Medical College Hospital, Kolkata. Out of 22 units screened for ABO blood group, volume, CBC, specific gravity, transfusion transmitted infection (TTI by ELISA) and HUCB culture by VITEK 2 system (Ramakrishna mission Seva Pratisthan,99 Sarat Bose Road, Kolkata, West Bengal) for aerobic instantly and anaerobic report obtained after 14 days of incubation. Among them 18 transfused human umbilical cord blood sample were analysed statistically (Table-1) and another unused 4 samples were discarded as per biological waste management protocol Government of India.

Table 1: Descriptive analysis of whole human cord blood.

Methods of Compatibility Testing

Blood grouping of samples both donor (HUCB) and Convalescent COVID-19 anaemic patients was done by tube methods using ABO and Rh antisera (Tulip) and each individual units were cross matched by column agglutination card (Bio-Rad).

Monitoring Transfusion and Oxygen Carrying Capacity.

18 patients with symptomatic anaemia (< 10 gm/dl), were transfused ABO and Rh matched, freshly collected screened (HIV, HBsAg, anti HCV, RPR and malaria) human umbilical cord blood (UCB) under supervision and followed up with complete blood count (CBC) for consecutive 4 months. Oxygen carrying capacity of everyone was assessed by RoHS CE, XECH, Japan and all data were recorded in master chart for statistical analysis [29,30].

Descriptive analysis along with quality control of human umbilical cord blood was done by analytical software Excel & IBM SPSS 20 and DATAtab online. Mathematical model is prepared to compare an equivalent quantity of adult human blood with 50% haematocrit versus cord blood in terms of haemoglobin and oxygen saturation [31]. Persistence of stability of post transfusion Hb level after cord blood versus adult human blood transfusion with 50% haematocrit effect was observed after 4 months (transfusion induced transplantation) [32,33].

Designing of Mathematical Model

• Minimum Hb is 12.5 gm/dl for voluntary blood donation [31]. The haematocrit (Hct) is about three times in respect of Hb gm/dl [34,35]. Haematocrit of 12.5 gm/dl is nearly 37.5%. 50% Hct contain nearly 16.67 gm/dl of Hb, (explanation – 37.5% Hct contain 12.5 gm/dl, 50% Hct contain 12.5X50/37,5=16.67 gm/dl).
• HUCB contain 14.4 gm/dl Hb of which 20% adult Hb (HbA) and 80% fetal Hb (HbF) [32,33].89 ml of HUCB contain 14.4X89/100= 12.8 gm/dl Hb (12.8X20/89=2.56 gm/dl HbA and 12.8X80/89=10.25gm/dl HbF , Total = 12.8 gm ). Oxygen carrying capacity of HUCB is 24.88 ml, (explanation 1 gm of adult Hb carries 1.39 ml of O2 and 1 gm fetal Hb carries 2.08 ml of O2, total oxygen carrying capacity 2.56X1.39= 3.56 ml of O2 and 10.25 X 2.08=21.32ml of = 24.88 ml).
• Adult Hb contain 16.67 gm Hb with 50% Hct and the oxygen carrying capacity of adult Hb is 16.67 X 1.39 = 23.17 ml, therefore, 89 ml of adult blood contain: 23.17X89/100= 20.62 ml of oxygen. Which is less than HUCB.

  Figure 1: Collection of human umbilical cord blood from screened pregnant mother.

  Figure: a) inspection of empty cord blood bag, b) spinal anaesthesia, c) surgical instrument required for caesarean section, d) surgical incision, e) steps of collection human umbilical cord blood, f) removal of placenta. Collection of human umbilical cord blood by Dr. Moinuddin Naskar, senior obstetrician of Vidyasagar Hospital Kolkata.

Altogether 18 convalescent COVID-19 anaemic patients (11 male and 7 female) were observed during 4-month study period. Pre haemoglobin level ranged from 7.1 gm/dl to 8.3 gm/dl, mean Hb 7.64 gm ± 0.37 gm. The profile of patients age, sex body weight distribution characteristics are given in Table- 2. All the 18 patients were followed up after 24 hours and 4 months of post human umbilical cord blood (HUCB) transfusion.

Table 2: Descriptive data of 18 patients given in table 2.

Assessment of Post- Transfusion Hb After 24 Hours And 4 Months

From Table -2, mean age, weight, gender, frequency of blood group, pre and post transfusion Hb in 24 hours and 4 months after transfusion was statistically analysed by using DATA tab online software.

The results of the descriptive statistics show that the pre- and post- human umbilical cord blood transfusion haemoglobin value (M = 7.64, SD = 0.37) and (M = 8.33, SD = 0.24), pre and post transfusion Hb given in figure 2 and figure 3. There was significant increment of Hb mean 0.69±0.24 gm in mean 66 kg of individuals by HUCB transfusion after 24 hours (p value < 0.01).

Figure 2: Line diagram of Hb pre and post.

Figure 3, Box plot of pre and post Hb.

Comparison of Post Transfusion Hb After 24 Hours and 4 Months

Post transfusion Hb of 18 COVID-19 positive patient was 8.33 gm/dl and 9.10 gm/dl in 24 hours and 4 months of human umbilical cord blood transfusion Figure- 4,5

Figure 4 & 5: shows box plot and line diagram of post transfusion Hb after 24 hours and 4 months. 

Comparison Between Male and Female Group

Comparative analysis among male and female HUCB transfusion recipients is given in the Venn Diagram Figure 6

Figure 6: Venn Diagram shows comparative analysis in between male and female.

From the above figure- 6, there was no statistically significant difference obtain in between male and female participants after HUCB transfusion [Figure-7,8], where the p value is > 0.05

Figure 7: Pre-transfusion Hb Vs Vol.

Figure 8: Gender wise Hb in pre and post Tran.

Above pictorial diagram shows volume of HUCB transfused with its effect in respect to Hb in male and female convalescent COVID-19 anaemic patients.

Mathematical Comparison in Between HUCB Versus Adult Red Cess with 50% Hct

From the above results a mathematical analysis for same quantity of Hb increment of in each individual patient with according to the body weight required more amount of adult blood (Hct 50%) Figure – 9

Figure 9: Line diagram.

A correlation of volume of HUCB and adult red cell volume in relation to post transfusion haemoglobin increment given in figure 9,

There was a significant difference (p < 0.001) shows in between HUCB (mean 89ml) and adult blood (mean 184 ml) requirement (50% hct ) for increment of 0.68 gm/dl Hb increment in mean 66 kg individuals. Comparative analysis of HUCB versus adult blood with 50% haematocrit done by Mann-Whitney U- test and mean rank and sum ranks of HUCB is 10 and 185 whereas mean rank and sum ranks of adult blood is 27 and 481. So, from above statistical calculation it shows that there was smaller quantity of HUCB required than adult blood with 50% hct which is statistically significant and p value <0.001.

Oxygen Carrying Capacity Comparison in Between HUCB and Adult Red Blood Cells with 50% Hct.

Mean oxygen carrying capacity of 184 ml adult blood is 31.76 ml (15.63-50.21ml), whereas oxygen carrying capacity of 89 ml HUCB is mean 24.88 ml (21.63-28.56 ml). comparative statistical analysis shows there was significant difference of oxygen carrying capacity in HUCB versus adult blood with 50% hct , p value is < 0.047 which is less than <0.05. Nonparametric statistical data is analysed by Mann-Whitney U test (mean rank and sum rank of HUCB and adult blood is 15, 270 and 22, 396) Figure – 10

Figure 10: Mathematical model of oxygen content in human red blood cells (50% hct) versus HUCB in 18 patients.

From figure 10 red line shows oxygen content in HUCB and green line shows oxygen content in adult red cells, mean 89 ml HUCB versus mean 184 ml adult red cells shows 24.88ml and 31.76 ml oxygen content.

Post Transfusion Hb After HUCB Transfusion

Bhattacharyya et.al. stated in the journal of American College of Surgeons in 2005 that placental umbilical cord whole blood transfusion is safe blood substitute in under resource country [36]. In the present study mean umbilical cord blood volume was 89 ml and mean haemoglobin was 14.4 gm%. Thus each 89 ml of human umbilical cord blood contained approximately 2.56 gm adult haemoglobin and 10.24 gm foetal haemoglobin that is a total of 12.81 gm haemoglobin {(14.4/100 X 89) X 20 = 2.56 gm and (14.4/100X89) X80=10.25 gm}. 89 ml human umbilical cord blood can carry 12.8 gm haemoglobin and 24.88 ml oxygen and directly it has the potential raise mean 0.68 ± 0.24 gm/dl haemoglobin in anaemic patients mean weight 66 kg and initiate improvement in the symptoms of anaemia in COVID-19 patients and this was statistically significant (p vale < 0.001). As per guidelines 2023, volume of transfusion required for rising 1 gm/dl of Hb with 50% haematocrit is 4.2 ml/kg [31]. Therefore, it seems to be assumed that increment of Hb 0.68 gm/dl in 66 kg a total 184 ml having 50% haematocrit (Hct) contain human blood transfusion is required. Comparative analysis HUCB versus adult red blood cells shows significant statistical differences by Mann Whitneys U test. Maria Bianchi et al. in Vox Sanguinis 2018 published a review of umbilical red blood transfusion in newborn patients with anaemia and concluded that HUCB might more physiological than adult red blood cells [37].

Transfusion Induced Transplantation.

Transfusion induced transplantation, also known as micro chimerism, can occur when cells from a transfused blood product, such as umbilical cord blood, are transferred to the recipient and engraft in the recipient’s tissues. This can lead to the development of a small population of cells in the recipient that have a different genetic makeup than their own cells, which is known as micro chimerism [38,39].

There was significant mean Hb rises 0.77 gm in post transfusion of HUCB (p value < 0.001) among the patients the mean weight was 66 kg. Figure-6 shows the sequential changes of Haemoglobin levels in same population.  Ebaugh FG et al., stated in their study that, survival of donor RBCs in recipients’ body is nearly 45 to 60 days [37]. Two studies were conducted by Luten et al., they observed that approximately 10-30% of the donor red cells are lost during post transfusion in the first 24 hours [40]. So, that despite decreases the mean Hb there was increasing the Hb in post transfusion of HUCB after 4 months without any additional medications (like haematinics) Figure-11. It is hypothesized (Figure-12) that the stem cells in umbilical cord blood, may engraft in the bone marrow of the recipient, and develop into new cells; this, in turn, might aid in the creation of red blood cells by the engrafted small population of hematopoietic stem cells [41,42,43]. However, the clinical significance of this phenomenon is not fully understood, and more research is needed to determine whether micro chimerism induced by umbilical cord blood transfusion has any therapeutic benefits.

Figure 11: Box plot of changes of Hb in post transfusion anaemic patients after 24 hours and 4 months.

Above figure shows the progressive changes of haemoglobin after freshly collected human umbilical cord blood transfusion.

Figure 12: Hypothetical understanding of how HUCB can correct anaemia.

Figure-12. is a pictoral depiction of how HUCB can potentially correct anaemia through a) transfusion induced transplantation or b) the impact of immunomodulatory substances present in whole HUCB, or c) erythropoietin secretion induced by HbF.

[HUCB- human umbilical cord blood, HbF -foetal haemoglobin, HSc-haematopoietic stem cells,MSCs – mesenchymal stem cells, EPO-erythropoietin, DC-dendritic cells, G-CSF-granulocyte -colony stimulating factors, VEGF-vascular endothelial growth factors,IGF-1-insulin like growth factors,TGF-β- transforming growth factor β, IL-8 -interleukin 8,IL-2 interleukin 2, IL6 interleukin -6, IL5-interleukin 5, IFN-γ interferon γ, TNF-α tumor necrosis factor α.] Diagrma by Dr.Biplabendu Talukdar

Comparative Analysis of HUCB with Whole Human Blood in Respect to Oxygen Carrying Capacity

Human umbilical cord blood contains foetal haemoglobin (HbF) and this was first noted by E. Von Korber in 1866 [44,45]. Foetal haemoglobin carries 50-60% more oxygen than adult haemoglobin [46], that is 1 gm of foetal haemoglobin may carry nearly 2.08 ml of oxygen whereas 1gm of adult haemoglobin binds with 1.39 ml of oxygen [47,48]. Bhattacharya et al. found that on an average human umbilical cord blood contain 20% adult haemoglobin and 80% foetal haemoglobin [49,50,51]. Total oxygen carrying capacity of 12.81 gm is 24.76 ml (adult haemoglobin carries 2.56 X1.39 ml=3.56 ml oxygen and foetal haemoglobin carries 10.2 X 2.08 ml= 21.32 ml oxygen, a total 3.56+ 21.32= 24.88 ml of oxygen). Whereas mathematical model shows 100 ml of adult red cells with 50% haematocrit may be able to carry 23.17 ml oxygen and 89 ml of adult red cells nearly 20.62 ml, which is less then oxygen carrying capacity of HUCB (100 ml adult red cells carrying 23.17 ml of oxygen, so 89 ml adult red cells carrying 23.17X89/100 = 20.67 ml). In hypoxemia old erythrocytes produced an organophosphate compound which is efficiently prevent tissue oxygenation. Transfused Fetal Hb stabilized the 2,3, DPG of adult Hb and initiate prevention of oxygen binding [52].

Human umbilical cord blood (HUCB) has been offered as a viable remedy to these drawbacks, which had been earlier transfused in various diseases without any immunological or non-immunological reactions. Using HUCB to treat anemia has been met with encouraging outcomes across several clinical trials. Fetal hemoglobin, anti-inflammatory cytokines, and growth factors are only some of the components of HUCB. Other components include hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, stromal stem cells, and stem cells like stem cells. HUCB is also considered to be safe for transfusion as the placental nano-barrier helps in protecting the blood from contamination in utero. These biochemical and cellular properties of HUCB can be utilized for the treatment of anemia.

Limitations of the study include small size of the sample patient in the experimental group. Secondly, HUCB was transfused only once in all the patients in the experimental arm.

Acknowledgement

The authors acknowledge for active support of Ms. Sanjukta Bhattacharya and Ms. Sanchayita Talukder in preparation of the manuscript and drawing of figures.

Conflict of Interest: Nil

Source of Fund: Nil

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