Comparative study of quality of leukoreduced packed red blood cell units as assessed by nageotte hemocytometry and flow cytometry.

PURPOSE: Assessment of residual white blood cell (rWBC) count is vital to ascertain the quality of leukodepleted (LD) blood components. Automated cell analyzers lack the sensitivity for the assessment of very few leukocytes as found in LD blood components. Flow Cytometry (FC) based methods and Nageotte hemocytometer are the most commonly used techniques for this purpose. The objective of this study was to compare the use of Nageotte hemocytometer and FC for quality control of LD red blood cell units. MATERIALS AND METHODS: A prospective, observational study was conducted in the Department of Immunohematology and Blood Transfusion of a tertiary care center from September 2018 to September 2020. About 303 LD-packed red blood cell units were tested by FC and Nageotte hemocytometer for rWBCs. RESULTS: The number of rWBC (mean) detected by flow cytometer and Nageotte's hemocytometer was 1.06 ± 0.43 white blood cell (WBC)/µL and 0.67 ± 0.39 WBC/µL, respectively. Coefficient of variation was 58.37% by Nageotte hemocytometer method and 40.46% by FC. Linear regression analysis did not show any correlation (R2= 0.098, P = 0.001) whereas Pearson's correlation coefficient showed a weak relation (r = 0.31) between the two methods. CONCLUSION: Flow cytometric technique provides a more precise and accurate objective tool compared to Nageotte hemocytometer which is labor intensive, time consuming, and prone to errors arising out of subjectivity along with reported underestimation bias. In the absence of adequate infrastructure, resources, and trained workforce, Nageotte hemocytometer method is a reliable alternative. Nageotte's chamber could be best used in the resource-constrained setup as it offers a relatively inexpensive, simple, and viable means to enumerate rWBCs.

A retrospective study to assess the impact of ABO incompatibility on outcomes of allogeneic peripheral blood stem cell transplants at a tertiary care hospital in Western Maharashtra.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) has emerged as a curative measure for life-threatening hematological disorders. It can be autologous or allogeneic depending on the disease characteristics. Providing transfusion support to the transplant patients can be challenging, especially in AB-mismatched allogeneic HSCT. In this study, we investigated the impact of ABO incompatibility in patients undergoing allogeneic HSCT. MATERIALS AND METHODS: A retrospective review was conducted in 76 patients with hematological diseases who underwent allogeneic HSCT. Transfusion requirements, engraftment profile, incidence of graft versus host disease (GvHD), and mortality for a period of 1 year were analyzed. RESULTS: ABO incompatibility between donor and the patient did not significantly affect the neutrophil and platelet (PLT) engraftment time (P = 0.389, 0.349, respectively), packed red blood cells transfusion requirement, and duration of initial hospital stay. However, patients of ABO-incompatible HSCT received more PLT transfusions posttransplant which was statistically significant. 29.1% of ABO compatible and 16.7% incompatible HSCT patients developed GVHD. Mortality rates in the two groups were 16.7% and 8.3%, respectively. However, differences in both the parameters were not statistically significant. CONCLUSION: Our study showed that ABO incompatibility does not significantly affect the outcome and should not be a limiting factor for selection of donor. Donor availability and human leukocyte antigen (HLA) matching remain the critical selection criteria.

A retrospective audit to evaluate the appropriateness and rationalization of Fresh Frozen Plasma usage in a tertiary care hospital.

Background: General guidelines describing the potential indications and contraindications of Fresh Frozen Plasma (FFP) exist. However, their implementation is inadequate, leading to inappropriate use in various clinical settings. This study aims to define the appropriateness of the FFP usage in terms of therapeutic versus prophylactic indications. Methods: Retrospective audit was conducted over one year prior to and after an educational intervention (1122 patients for 6072 FFP units and 1061 patients for 4858 FFP units, respectively). Clinical diagnosis, indication for FFP transfusion, and coagulation profile were noted, and episodes of transfusion were divided into appropriate and inappropriate based on the guidelines of the British Committee for Standards in Hematology (2004 reviewed in 2018) and College of American Pathologists (1994). Results: Initial audit found 51.8% of FFP transfusions to be inappropriate (3069 of 5922). Coagulation profile (with INR values less than 1.5 times of the normal) was the most common cause of inappropriate transfusion (15.08%). 56.7% of FFP were prophylactically transfused. Re-audit after educational interventions showed a 22.3% reduction in the number of inappropriate transfusions. Conclusion: Inappropriate, as well as high prophylactic usage of FFP, was noticed in the initial audit, which reduced significantly after educational interventions. Regular CMEs, interactive sessions with clinicians, functioning Hospital Transfusion Committees, and prospective audits can affirm, further improve and reinforce the existing Hospital Transfusion Guidelines.

Association of CD 34 positive cell dose with engraftment kinetics in autologous peripheral blood stem cell transplant patients of multiple myeloma.

Background: Treatment with high-dose chemotherapy and stem cell transplantation has prolonged survival in patients of multiple myeloma (MM). A dose-response relationship between number of CD34+ cells infused and leukocyte and platelet recovery, exists. Patients receiving dose of <2.0 × 106 CD34+ cells/kg have delayed engraftment. The level of optimal cutoff for accelerated engraftment is yet to be validated. Hence, this study was undertaken to study the association of CD 34+ cell dose with engraftment kinetics in patients of MM who underwent autolgous peripheral blood stem cell transplant (PBSCT). Methods: We retrospectively analyzed 19 patients of MM who underwent PBSCT at our center between December 2016 to December 2018. Complete blood counts were carried out daily after transplantation to record neutrophil and platelet engraftment. Results: Based on the CD34+ cell dose given : <5 × 106/kg (category 1), 5-10 × 106/kg (category 2), >5 × 106/kg (category 3), the mean (SD) neutrophil engraftment time was 11.3 (0.5) days, 10.6 (0.9) days, and 10.2 (1.3) days respectively. Platelet engraftment time was 12.4 (2.60) days, 10.6 (1.14) days, and 11.2 (1.64) days for category 1, 2, and 3 patients, respectively. Correlation co-efficient between CD 34+cell dose and days for neutrophil and platelet engraftment was found to be -0.24 and -0.20, respectively. Time for neutrophil engraftment was found to be significantly associated with CD34+ cell dose category. Conclusion: CD 34+ cell dose appears as the strongest predictor of leukocyte and platelet engraftment. CD 34+ cell dose of >5.0 × 106 cells/kg leads to an accelerated neutrophil and platelet engraftment in patients of MM.

A study to assess the relationship between donor uric acid levels and supernatant hemolysis in stored packed red blood cell units.

BACKGROUND: Most of the red blood cell (RBC) storage lesions can be attributed to oxidative stress encountered by the RBCs throughout the duration of their storage. Various donor variables at the time of donation may be responsible for the total antioxidant capacity of the supernatant and thus, the "storability" and the magnitude of development of these RBC storage lesions. It is known that uric acid (UA) is responsible for more than 60% of the TAC of the blood. This study aims to explore the relationship between donor UA levels and the difference in percentage hemolysis, an important RBC storage lesion, on day 1 and day 21, in stored packed RBCs (PRBCs) units. MATERIALS AND METHODS: The serum UA of 100 healthy voluntary male blood donors was estimated at the time of blood donation. The percentage hemolysis in the supernatant of the leukoreduced citrate phosphate dextrose/saline-adenine-glucose-mannitol RBC units (n = 100) prepared from these donors was calculated on day 1 and day 21. The difference in percentage hemolysis between donors with high normal serum UA levels (>7 mg/dL) was compared to that of the donors with low normal serum UA levels (<5 mg/dL) to observe the effect of donor UA levels on the difference in percentage hemolysis. RESULTS: The mean of the differences in percentage hemolysis in the supernatant in low UA group (<5 mg/dL) was higher than the mean of the differences in percentage hemolysis in the supernatant in high UA group (>7 mg/dL) and this was statistically significant (P < 0.001). The donor serum UA level and difference in percentage hemolysis on day 21 and day 1 were found to be negatively co-related. CONCLUSION: Higher levels of serum UA of blood donors seem to have a protective effect on the stored PRBC units as shown in this study. Hence, the potential of UA as one of the constituents of RBC additive solutions might lead to the enhancement of the quality of stored PRBC units by decreasing the RBC storage lesions.

Effect of prolonged storage at 2°C-6°C for 120 h on the coagulation factors of thawed cryoprecipitate: Can we extend its shelf life post thaw beyond 4 h?

BACKGROUND: Cryoprecipitate helps in replenishing important coagulation factors like fibrinogen, Factor VIII and von Willebrand factor without running the risk of volume overload. It is very useful in the treatment of trauma patients with active bleeding and works best when administered early. Extending the shelf life of thawed cryoprecipitate beyond 4 hours enables us to manage inventory better, reduces the burden of demand vs supply as well as minimizes wastage. It can also help in logistically supporting the transfusion services in making cryoprecipitate readily available in mass casualty scenarios (war, natural calamity) in remote locations by reducing the time required for thawing cryoprecipitate and the need for costly storage equipment. AIM: The aim of this study was to compare the levels of Factor VIII, Fibrinogen and von Willebrand factor on thawed cryoprecipitate after prolonged storage for 5 days at a temperature of 2-6°C. METHODOLOGY: The above mentioned coagulation factors were analyzed in cryoprecipitate at the time of product thaw and again after 120 hours of 2 to 6°C storage using fully automated coagulation analyser (STA Compact Max). All parameters were expressed as Mean ± Standard deviation and were analyzed using paired t-test with level of significance, P < 0.05. RESULTS: There was a significant decrease in the level of Factor VIII, whereas the levels of fibrinogen and von Willebrand Factor remained stable during the storage period. All the cryoprecipitate units retained factor activities above therapeutic range even after 5 days of storage at 2-6°C. CONCLUSION: Although the levels of clotting factors are reduced during storage, they are still maintained above the therapeutic range. In scenarios where maintaining frozen cryoprecipitate inventory is a logistical challenge and emergency massive demands of cryoprecipitate are foreseen, the use of pre-thawed cryoprecipitate can be considered as a viable option.

Allogeneic Peripheral Blood Stem Cell Transplant: Correlation of Donor Factors with Yield, Engraftment, Chimerism, and Outcome: Retrospective Review of a Single Institute During a 3-Year Period.

BACKGROUND: Donor factors have a variable correlation with cluster of differentiation (CD)34+ cell dose in allogeneic peripheral blood stem cell (PBSC) harvests. CD34+ cell dose affects the speed of hematopoietic recovery and percentage of donor chimerism in the recipient. METHODS: A total of 25 allogeneic PBSC transplants performed during a 3-year period were included. All donors underwent mobilization with filgrastim. Leukapheresis, flowcytometric CD34+ cell enumeration, and chimerism analysis were performed and correlated with recipient outcome. RESULTS: Besides age, all other donor parameters had a positive correlation with CD34+ cell count. Engraftment kinetics and chimerism had a positive correlation with the CD34+ yield of the PBSC product. Acute graft-vs-host disease (GVHD) was observed in patients with complete chimerism at day 30 after transplantation. CONCLUSION: Adequate CD34+ cell yield happens in healthy donors, independent of donor demographic patterns with G-CSF only. A diverse population of donors can thus be approached for Matched Unrelated Donor (MUD) transplants. An accurate quantitative analysis of early donor chimerism in the recipient (at day 30) is an excellent tool for post-transplant monitoring for acute GvHD.

Successful therapeutic plasma exchange in a patient with Morvan syndrome.

Morvan syndrome is a rare autoimmune disorder, characterized by hyperexcitability of both central and peripheral nervous systems, accompanied by autonomic dysfunction and hallucinations.[1] Therapeutic plasma exchange (TPE) has been found to be an effective mode of treatment for this disease, but there is limited literature supporting the same.[2] A 26-year-old male was admitted to our hospital and diagnosed with a case of Morvan syndrome, based on the clinical picture and laboratory findings. When standard drug therapy failed to show any improvement, a decision to carry out TPE was taken. The case presented with many peculiar challenges, mostly due to autonomic instability and hyperkinesia experienced by the patient while carrying out the procedure. All these challenges were diligently addressed and managed promptly. Clinical signs of improvement were evident from the 2nd TPE and by the time fifth TPE had finished, the patient was able to perform activities such as walking with support. His autonomic dysfunction and behavioral abnormalities had significantly subsided. This case report highlights the possible effectiveness of TPE in the management of a rare disease such as Morvan syndrome and appropriate application of basic principles and criteria for the use of TPE in cases where limited literature is available.

Point of care hemoglobin testing in plateletpheresis donors: Noninvasive versus invasive methods.

BACKGROUND: Predonation hemoglobin (Hb) for plateletpheresis donors is estimated by presently available invasive methods. Venous samples of only those donors whose Hb is more than 12.5 g/dl are screened for complete blood count and transfusion transmissible infections. There is a pressing need to substitute this invasive Hb test with noninvasive one to reduce donor discomfort and avoid further pricking the donor. We therefore went ahead with the aim of comparing a noninvasive Hb estimation method NBM 200 with the invasive method - Hemocue, taking SYSMEX KX-21 as a gold standard. METHODS: 500 voluntary consenting plateletpheresis donors qualifying the laid down criteria for platelet donation were included in the study. Hb readings obtained by the NBM 200 and Hemocue were compared to those obtained from the fully automated hematology analyzer SYSMEX KX-21. RESULTS: Coefficients of correlation were found to be statistically significant at <0.0001 level of confidence. Results of Friedman's test on the three methods also showed significant difference in means. Bland-Altman plots and mountain plots also confirm the same. NBM 200 was found to be more sensitive, specific, and precise than Hemocue in detecting ineligible donors. CONCLUSION: NBM 200 was found to be more sensitive, specific, and precise as compared to Hemocue for predonation screening of Hb in plateletpheresis donors and the prime benefit it offers is that it is 'noninvasive' thereby assisting in stemming the platelet donor pool. The onus lies on the blood transfusion services to make use of appropriately validated gadgets that reduce the donor discomfort.

Antiphospholipid syndrome: A diagnostic challenge.

The antiphospholipid syndrome (APS) is an acquired autoimmune thrombophilic disorder that is characterized by thrombosis (venous, arterial and microvascular) and obstetric morbidity due to a diverse family of antibodies against phospholipid-binding proteins present in plasma. The term antiphospholipid antibody is actually a misnomer as the antibodies are not against the phospholipid per se, but target the plasma protein co-factors, which bind to anionic PLs. The exact etiology has not been elucidated and is multifactorial. The initial guidelines for the diagnosis of APS were laid down in Sapporo, 1999, which were subsequently revised as the Sydney Consensus Conference criteria in 2006. Major changes were the inclusion of ß2GPI as independent laboratory criteria, addition of ischemic stroke and transient cerebral ischemia as established clinical criteria and the requirement of repeating the test after 12 weeks. The laboratory tests recommended are coagulation assays, which study the effect of lupus anticoagulant on the clotting time and immunological assays, mostly ELISAs to detect IgG and IgM antibodies against cardiolipin and/or ß2 glycoprotein I. For the diagnosis of APS, at least one clinical criterion and one laboratory criterion should be present. Limitations pertaining to the standardization, reproducibility and robustness of the currently recommended diagnostic tests still remain. Despite elaborate guidelines and syndrome defining criteria, the diagnosis of APS still remains a challenge. A greater interaction between the clinicians and the laboratory professionals is necessary for arriving at the correct diagnosis as a misdiagnosis of APS can have grave consequences.

Optimizing cord blood collections: Assessing the role of maternal and neonatal factors.

BACKGROUND: As processing and cryopreservation of cord blood is time consuming and costly, it is essential to select units with optimal CD34+ cells, total nucleated cell (TNC) number and colony forming units (CFUs). These are the most important factors affecting outcome of UCB transplantation and are influenced by various maternal and neonatal factors. AIM AND OBJECTIVES: To determine the maternal and neonatal factors affecting TNC and CD34+ cell counts in cord blood so as to aid in proper selection of cord blood units for cryopreservation. MATERIALS AND METHODS: A total of 100 UCB units were collected from normal vaginal deliveries, processed and assessed for volume, TNC, CD34+ cell count and CFU-GM. These parameters were then analyzed to find out whether they correlated with maternal and neonatal characteristics such as mother's age, parity, gestational age, baby's birth weight, and sex. RESULTS: The volume of CB collected significantly correlated with the TNC, CD34+ cell, and CFU-GM yields (P < 0.02). A heavier placenta (P < 0.05), and a heavier baby (P < 0.002) were associated with a significantly greater volume of CB whereas the age, parity of mother and the sex of the baby had no significant effect. CONCLUSION: The only factors found to affect the TNC and CD34+ cell counts significantly were weight of the baby and placenta and the volume of cord blood collected. Since these factors are of prognostic significance, their analysis will aid in deciding which UCB unit should be processed and cryopreserved for UCB banking and subsequent transplantation.

Report of two cases of anti-M antibody in antenatal patients.

Anti-M is a relatively common naturally occurring antibody reacting optimally at 4°C and weakly or nonreactive at 37°C. It is usually clinically insignificant but can be active at 37°C because of thermal amplitude of IgM component or presence of IgG component. It can cause or delayed hemolytic transfusion reactions or hemolytic disease of newborn. At our center we have encountered two cases of anti-M antibodies- one presenting as crossmatch incompatibility and other as blood grouping discrepancy in the last 8 months.

Red blood cell alloimmunization in multitransfused patients in a tertiary care center in Western India.

OBJECTIVE: To investigate the seroprevalence and specificity of red blood cell (RBC) antibodies in multitransfused patients, in whom the risk of alloimmunization is especially high. METHODS: We conducted a retrospective study on blood specimens from 200 multitransfused patients. We evaluated all specimens for alloimmunization using various immunohematological tests via the column agglutination technique. RESULTS: The overall prevalence of RBC alloantibodies was 5.5%. Of the 11 specific types of alloantibodies identified, most (72.7%) belonged to the Rh blood group system, followed by the S, M, and Lewis blood group systems (9.1% each). CONCLUSION: Most alloantibodies were of the Rh blood group specificity. To improve the quality of blood supplied, especially to patients with thalassemia, we recommend that Rh phenotyped, cross-match-compatible blood should be issued to prevent complications such as acute and delayed hemolytic reactions.

Microcytic hypochromic anemia: should high performance liquid chromatography be used routinely for screening anemic and antenatal patients?

BACKGROUND: Hemoglobinopathies are the most common inherited red cell disorders worldwide. Identification of these disorders is immensely important epidemiologically and for improved management protocols. AIM AND OBJECTIVES: Our aim was to determine the prevalence of hemoglobinopathies in patients with microcytic hypochromic anemia and to assess the suitability of using high performance liquid chromatography (HPLC) routinely for screening antenatal cases and patients with anemia. MATERIALS AND METHODS: A total of 4335 cases received from Mar 2007 to Nov 2011 were studied for various hemoglobinopathies and variants on BIO RAD 'VARIANT' analyzer. RESULTS: Of the 4335 cases studied, 2119 were antenatal cases, 1710 patients with other disorders and 506 family studies. Of these, 688 cases displayed abnormal hemoglobin fractions on HPLC of which 140 were antenatal women. There were 455 cases of ß thalassemia trait, 24 ß thalassemia major, 20 thalassemia inter-media, 54 sickle cell trait, fivesickle cell disease, 21 double heterozygous ß thalassemia-sickle cell trait, nineand 4 Hb D- Punjab heterozygous and homozygous respectively, three Hb D ß Thalassemia trait, 20 and 37 Hb E homozygous and heterozygous respectively, three Hb E ß Thalassemia trait and four cases of Hb Q India. Twenty nine adults had isolated HbF elevation. CONCLUSION: Our study found a high prevalence (15.8%) of hemoglobinopathies amongst microcytic hypochromic anemia and antenatal cases. An accurate diagnosis helps in preventing unnecessary iron loading. Screening all antenatal cases with anemia helps in timely antenatal counseling, thus preventing the psychological trauma of bearing a transfusion dependent child for life.