Expectations Vs Achievements - Quality Control Of Blood Components Sitalakshmi S Professor and Head Department of Clinical Pathology St John’s Medical College, Bangalore

The Case……. A 65-year old lady, was referred to the Medical College Hospital for recent onset fatigue and weight loss . A CBC showed a WBC count 55,000/ul with 90 % peroxidase positive blasts, normochromic normocytic anemia (Hgb 8 g/dl) and thrombocytopenia (40,000/ul).

On physical exam, the patient was afebrile, normotensive There were no bleeding manifestations An admission ECG and history are negative for cardiovascular disease

Need for transfusion? Does the patient require transfusion of: packed red blood cells? platelets? Although the patient is mildly anemic and thrombocytopenic, she does not fulfill criteria for transfusion at this time. Her Hgb > 7g/dl with no history of cardiovascular disease or symptomatic anemia (other than fatigue).

If the patient had a history of cardiovascular illness or signs or symptoms of significant anemia, it would be appropriate to maintain her Hct at a higher baseline. Similarly, the patients platelet count (40,000/ul ) with no bleeding manifestations does not require prophylactic platelet transfusion

Further Clinical Course…. Eight days after admission for induction chemotherapy, the patient's CBC shows: WBC 500/ul Hgb 6.9g/dl Platelet 8,000/ul The patient's physician ordered 2 units leukoreduced and irradiated packed red blood cells and 6 units of pooled platelet concentrates (3 X 10¹¹ platelets total). Was transfusion of PRBC and platelets appropriate in this patient at this time?

Need for transfusion….. Yes. The patient's hemoglobin < 7.0 g/dL in the setting of a bone marrow failure Likewise, the patient's platelet count has now decreased to the point where she is at risk for spontaneous hemorrhage

A CBC 1 hour after the administration of platelets and PRBC shows: Hgb 9.1g/dl Hct 27.3 % Platelets 30,000/ul Corrected count increment (CCI) (postTPC - preTPC) x (BSA in M²)/(TNPTx 10¹¹) where: TPC=transfusion platelet count BSA=body surface area TNPT=total number of platelets transfused For this patient: post TPC 30,000 pre TPC 8,000 CCI=(30-8) x 1.5/3 = 11

Did the patient get an appropriate response from the PRBC and platelet transfusions? Yes to both. The patient was transfused with 2 units of packed red cells (PRBC), Hgb of 6.9g/dl rising to 9.1g/dl. An average adult show a 1 g or 3% rise in Hct with each unit of packed red cells. Likewise, the calculated CCI was 11.0. A CCI of > 7.0 is considered a good clinical response.

What are the factors that affect the patient’s response to transfusion of blood components?

Quality of blood components transfused Patient’s disease condition and patients physiological response to transfusion How do we as transfusion specialists ensure quality of blood components? What is our role in patient transfusion therapy?

Quality programmes and systems in transfusion medicine differ slightly from other disciplines of laboratory medicine It takes two different forms. the blood supply system and Clinical transfusion therapy.

The approach used to ensure highest quality in the blood supply system is similar to that used in the manufacture of pharmaceuticals The approach used to ensure that the patient receives highest quality of transfusion therapy is a part of hospital’s overall quality care program

The measures taken for quality assurance in transfusion medicine are: Measures needed during the process of blood component preparation and storage Measures taken during the process of actual transfusion of the blood components. This aspect has more to do with good clinical transfusion practices

Standards for Quality monitoring The wide variability of the source material from which blood components are prepared makes it difficult to set stringent limits. Nevertheless, realistic minimum specifications should be set and complied with.

Standards for quality monitoring The standards are based on minimum requirements On what is achievable using the most up-to-date methods and systems Testing of random components to ensure they achieve reliably minimum specific requirements as per the standards

Component preparation The quality of the components is assured by control of all stages of manufacture donor selection Blood collection Component separation labeling Storage

The starting materials for preparation of blood components are blood donations collected from suitable donors. Conditions of storage or transport and the time prior to processing, are contributing factors to the quality of the product.

Delays in preparation unsuitable conditions of storage or transport may adversely affect the quality of the final product. Blood and blood components should be placed in controlled and validated conditions as soon as possible after collection.

Quality assurance is concerned with every aspect of transfusion practice and applied to all activities of blood transfusion service from: QUALITY ASSURANCE Identification of suitable Blood donors Collection of blood Preparation of blood components Blood transfusion

Srandard Operating procedures The standard operating procedures should describe the specifications for materials that will influence the quality of the final blood component. Specifications should be in place for blood components (intermediate and final components), starting materials, additive solutions, blood collection bags and equipment.

Blood Collection The collection process itself is crucial for the quality of blood components. Measures such as a reliable arm-cleaning and disinfection procedure, the use of closed and sterile collection systems should be implemented. Free flow of blood during collection is crucial Time limits is defined for the processing of blood components.

Each component should be visually inspected at each stage of processing and immediately prior to issue. The component must be withdrawn if there is evidence of leakage, damage to or fault in the container, excessive air, suspicion of microbial contamination or any other contraindications such as platelet clumping, unusual turbidity, haemolysis or other abnormal colour change.

Frequency of quality control Should be performed on at least 1% of all components produced for all parameters to be measured If fewer than 100, then at least 4 per month 75% or more of components monitored must meet the specifications

Transport Donations and samples should be transported to the processing site in accordance with procedures that ensure a constant approved temperature. This is especially important when blood is transported from distant collection sites. Portable temperature loggers may be used to record the temperature during the transportation of blood to the processing site.

Component weight: volume To provide information, which is useful for clinicians, the component specifications generally require the component label to indicate a volume. whole blood volume is calculated by deducting the weight of the bag assembly and dividing the resulting weight by the specific gravity of 1.06

Component weight: volume For red cell components, volume is calculated by weighing the pack, deducting the weight of the pack assembly only and dividing the resultant weight by the specific gravity 1.09 For platelets and plasma components, volume is calculated by weighing the pack, deducting the weight of the pack assembly and dividing the resulting weight by the specific gravity of 1.03.

Standard Operating procedures The standard operating procedures for component preparation should be followed at all times using the validated methods. Any deviations from these established procedures and processes may result in products not meeting specifications and such products should be considered as non-conforming products and must not be released .

Methods of production Blood components may be prepared by using a centrifugation step with subsequent separation, or by apheresis technology during collection The use of closed systems is a must for all steps in component processing. Where sterile connecting devices are used to maintain a functionally closed system they should be correctly used in accordance with a validated procedure. The resulting weld should be checked for satisfactory alignment and integrity.

Equipment Equipment used in collection, processing, testing and storage of blood and blood component should be observed, standardized and calibrated on a regularly scheduled basis Procedures should be described in the Standard Operating Procedure (SOP) Manual

Equipment performance monitoring

Refrigerated centrifuge Calibrated upon installation and repair Calibration of speed with tachometer twice a year Timer to be checked with tachometer

Platelet agitator Number of strokes 70+/- 10 per min Periodic cleaning and lubrication

Quality Control of equipment for component storage Daily periodic temperature monitoring of refrigerators, freezers and platelet incubator Temperature check at different locations in large equipment Audio and visual alarms to alert Continuous temperature recorder Actual temperature checked with Hg themometer in glycerol

centrifugation The centrifugation parameters (revolutions per minute, temperature, time, acceleration, deceleration) are important for the composition and characteristics of the specific components. These critical parameters should be defined on the basis of validation data that demonstrate a process that consistently produces quality products. For each run, the centrifugation records should identify the operator and confirm that the centrifugation process was performed according to specifications.

Separation After centrifugation, the bag system should be carefully removed from the centrifuge and placed into a plasma expressor or blood separation system. The different layers of the components (red cells, platelets, plasma) should be transferred to the satellite bags within the closed systems, in a manner designed to optimize the harvest of the intended component while minimizing the carry-over of other component fractions. Alternatively, blood components can be separated during collection by apheresis technology

Packed Red cell concentrate Red-cell concentrates are obtained from whole blood by centrifugation and removal of plasma with or without buffy coat, depending on the centrifugation parameters. The red cells should be stored at 2–6°C as soon as possible. Alternatively, red-cell concentrates may be obtained using an apheresis system and stored at 2–6°C.

leucoreduction To obtain leukocyte-reduced red-cell concentrates, either whole blood filtration can be applied prior to separation or there can be a post-separation filtration of the red-cell concentrate.. Red-cell concentrates are stored at 2 -8oC. The storage time depends on the anticoagulant/preservative solution used. The introduction of any leukocyte reduction process either by filtration or special centrifugation technique requires careful validation

leucoreduction Counting residual WBC by automated cell counters may not be reliable for very low WBC count of < 100WBC /ul Nageotte counting chambers are reliable for as low as 1 WBC/ul Flow cytometer can detect as low as 0.1 WBC/ul

Qc of red cell concentrate PARAMETER QUALITY REQUIRMENTS Frequency of testing VOLUME 280+/-60 ML 1% of all units HEMATOCRIT 0.70+/-0.05[ >70%] Sterility Culture

Further methods of preparation, such as irradiation or washing, are applied to obtain specific red-cell products, depending on the clinical indication. Periodic quality control should be performed on the final product to ensure that the manufacturing process is consistent At a minimum, the following critical parameters should be checked during the quality control assays: — volume — haematocrit — haemolysis at the end of storage — residual leukocytes, if leukocyte reduction is performed.

Fresh frozen plasma

Freezing of plasma Freezing is an important processing step that has an impact on quality of plasma. The rate at which freezing proceeds and the core temperature are both important parameters. Freezing speed will be influenced by the the freezing equipment, loading pattern and the volume of plasma. Rapid plasma freezing prevents or reduces the loss of critical constituents such as Factor VIII in frozen plasma.

Fresh Frozen Plasma FFO is prepared either from whole blood or from plasma collected by apheresis, and is frozen within a defined period of time to a temperature that should adequately maintain the labile coagulation factors in a functional state. Factor VIII content is critical both as a quality indicator and to assure the efficacy of cryoprecipitate. If plasma is separated from a unit of whole blood that is refrigerated to 4°C, centrifugation should preferably take place within eight hours of collection

QC OF FFP PARAMETER QUALITY REQUIRMENTS Frequency of testing VOLUME 200-250 ML 4 units per month or 1% STABLE COAGULATION FACTORS 200 UNITS FACTORVIII 0.7 UNITS/ML FIBRINOGEN 200-400 mg

Platelet concentrate

Platelet concentrates Platelet concentrates are derived from whole blood or are obtained by apheresis. After collection, whole blood can be kept at 20°C and 24°C,. for up to 4 – 6 hours The whole blood unit is centrifuged so that an optimal number of platelets remain in plasma (platelet-rich plasma, or PRP). Platelet concentrates are then obtained by hard-spin centrifugation of PRP and are then resuspended.

Platelet concentrates if whole blood is centrifuged so that the blood platelets are primarily sedimented to the buffy coat layer, the buffy coat is separated and further processed to obtain a platelet concentrate. Either a single buffy coat or a pool of buffy coats is diluted with plasma, and platelets are concentrated by further centrifugation. The platelet content per unit depends on the method of preparation. Similarly, the residual leukocyte content will vary according to the centrifugation parameters

Platelet concentrates (both from whole blood and apheresis) should be stored in conditions that guarantee that viability and haemostatic activities are optimally preserved. The storage temperature should be 20–24°C. Continuous gentle agitation of platelets during storage should be sufficient to guarantee the availability of oxygen to the platelets (but should be as gentle as possible). A storage time should not exceed five days

QC of platelet concentrate (prepared from buffy coat) Parameter Quality requirement Frequency of control Volume 70-90 ml 4 units / month Platelet count > 6-9 x 1010 pH > 6.0 RBC contamination Traces to 0.5 ml WBC contamination < 5.5 x 106

QC OF PLATELET CONCENTRATE by PRP PARAMETER QUALITY REQUIRMENTS Frequency of testing VOLUME 50-70ML All units PLATELET COUNT >3X10 10 / UNIT 4 units per month or 1% if > 100 PH >6 RBC CONTAMINATION <0.1X10 12/L WBC CONTAMINATION <1.5X10 9/L

QC OF PLaTelet concentrate BY APHERESIS PARAMETERS QUALITY REQUIRMENTS VOLUME 200-500 ml PLATELET COUNT >3.0X10 11/unit PH >6 LEUCOCYTES < 0.03X10 9/L RED CELLS TO 0.5ML

Calculation the platelet count per bag Weight of empty bag=24gm Volume =weight of plt bag—wt of empty bag 1.03 Eg:Wt OF PLT BAG=89 VOLUME=89-24=65gm,TO CONVERT TO mL 65 =63ml

VOLUME=63ml PLT COUNT=NO.OF PLT/ulX1000XVOLUME PLT COUNT=10X1000X63 ==6.3 X1010/L

Cryoprecipitate Cryoprecipitate is the cryoglobulin fraction of plasma and contains a major portion of the Factor VIII, von Willebrand factor, fi brinogen, Factor XIII and fibronectin present in plasma.

Cryoprecipitate Plasma is allowed to thaw either overnight at 2–6°C or by a rapid-thaw technique. Following thawing, the supernatant cryo- poor plasma and the cryoprecipitate are separated by hard-spin centrifugation. The cryo-poor plasma is then expressed into a transfer bag. The two components are refrozen to the appropriate core temperature.

QC OF CRYOPRECIPITATE PARAMETER QUALITY REQUIRMENTS VOLUME 200-250 ML PLASMA STABLE COAGULATION FACTORS 200 UNITS FACTORVIII 0.7 UNITS/ML FIBRINOGEN 150-250 mg

Qc of cryoprecipitate FOR FACTOR VIII assay -TAKE 1/10 DILUTION [0.9ml IMIDAZOLE& 0.1ml CRYO] % ACTIVITYX DILUTION FACTORX VOLUME OF CRYO Eg:96X10X12mL = 111.20IU/BAG 100

Ensuring quality of the prepared blood components is the first step towards achieving optimum benefit to the patients Ensuring good clinical transfusion practices is of utmost importance for optimum benefit to patients

Clinical Transfusion Process The Transfusion of the right blood component to the right patient at the right time, in the right condition and according to appropriate guidelines A chain of integrated events that begins with a correct decision that the patient needs blood and ends with an assessment of the clinical outcome of the transfusion. Its goal is to achieve optimal use of blood

Optimal use of blood The safe, clinically effective and efficient use of donated human blood Safe: No adverse reactions or infections Clinically effective: Benefits the patient Efficient: No unnecessary transfusions Transfusion at the time the patient needs it

AABB seeks to promote an interdisciplinary dialog that recognizes the critical importance of blood transfusion while also encouraging an approach to patient care that is designed to reduce the occurrence of preventable transfusions. Such an approach requires the involvement of clinicians, laboratory personnel, nursing staff, surgeons, anesthesiologists, and administrators.

Quality transfusion services Clinical transfusion guidelines audit systems to monitor adherence to the guidelines the hemovigilance program which monitors the entire blood supply value chain,

Each of these systems has a different focus: the aim of clinical transfusion guidelines is to give direction to the clinician on when to transfuse and what the expected outcome should be. Guidelines are therefore a tool to ensure appropriate utilization of blood products. This is of critical importance in light of possible transfusion complications and a dwindling donor base.

Each patient must be evaluated individually and, if justified, the clinician can adjust the treatment guidelines for a specific patient. The decision to transfuse is therefore based ultimately on a clinical assessment of a specific patient’s condition and appropriate laboratory parameters. Transfusion therapy must always be of overall benefit to the patient.

In the field of transfusion practice, controversies exist in the following areas - When to transfuse? What to transfuse? How much to transfuse?

QUESTIONS TO BE ANSWERED IN THE DECISION MAKING PROCESS

What improvement in the patient’s clinical condition am I aiming to achieve? Can I minimize blood loss to reduce this patient’s need for transfusion? What are the specific clinical or laboratory indications for transfusion for this patient? What is the time frame of decision making urgent/elective?

What are the risks of acquiring HIV/ hepatitis, through the blood products that are available for this patient? Do the benefits of transfusion outweigh the risks for this particular patient? How should the component be administered, monitored and its efficacy documented? Is the component easily available? What other options are there if no blood is available in time?

Will a trained person monitor this patient and respond immediately if any acute transfusion reactions occur? Have I recorded my decision and reasons for transfusion on the patient’s chart and the blood request form? Is the patient/relative fully informed and consenting to medical decisions?

The primary goal of modern blood transfusion therapy is to ensure that the patients receive safe, reliable and effective transfusion of the required blood components where the benefits outweigh the risks. The transfusion medicine specialists plays an important role in guiding the clinicians regarding good transfusion practices and also ensure preparation of blood components as per specifications of standards

tHank you