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Indian Journal of Transfusion Medicine
  Indian Journal of Transfusion Medicine Indian Journal of Transfusion Medicine

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Internal Quality Control In Blood Bank Testing

 

Dr. Rohit Jain,

Dr. Vinod Sharma,

Dr. G. N. Gupta

Internal Quality Control (IQC) is the backbone of  any quality assurance program. Internal quality control is the set of procedures undertaken by the staff of a laboratory for continuously and concurrently assessing laboratory work and the emergent results, to decide whether they are reliable enough to be released. It is meant to allow laboratory technicians to check their own performance and help them to monitor the reliability of their technique. It maintains day to day working objectively and detects any deviation very early like

Drift or trend - Seen when the control value moves  progressively in one direction from the mean for a minimum of three (3) days. Suggests that a problem is gradually developing, such as deterioration of a reagent or control.

Dispersion – Increase in Random Errors and Lack of Precision. Suggests inconsistency of technique or fluctuations in instrumentation function.

Shift – Abrupt changes observed when a problem  develops suddenly. May be due to instrument malfunction or an error in technique.

QUALITY MANAGEMENT in blood transfusion service is concerned with every aspect of transfusion practice and applies to all activities of a blood transfusion service. It involves identification and election of prospective blood donors, adequate  ollection of blood, preparation of blood components, quality laboratory testing and ensuring the safest and most appropriate use of blood/blood components.
When testing of blood & its components is performed, test may either be classified as:

QUANTITATIVE: Measures the amount of substance present like in TTD (ELISA,MILUMINESCENCE), Final products and Donor selection parameters.

 QUALITATIVE: Determines whether the substance being tested is present or absent like in Blood  rouping, rapid card tests etc.Internal quality control programme try to make things more objective than subjective and to reduce interpersonal or inter organization errors/differences.

 
List of Qualitaive(QL) and Quantitaive (QN) tests done in blood bank
 
 TESTS FOR DONOR  ELECTION

TESTS OF COLLECTED BLOO PRODUCT

TEST OF FINAL PRE TRANSFUSION SEROLOGICAL TESTS DONE
Hemoglobin (QN)       
HIV(QN)/(QL-CARD TEST)                                                
Hemoglobin (QN)                     
Bloodgrouping (QL)
Platelet count (QN)    
HBs Ag (QN)/ (QL-CARD TEST)                                              
PCV(QN)                         
Cross Matching (QL)
SerumAlbumin  
HCV (QN)/ (QL-CARD TEST)                        
Platelet count (QN)                         
Antibody screening / (plasma identification (QL)
Donors) (QN)      
  VDRL/RPR(QL) pH (QN) Direct Coombs Test (QL)
  MP (QL) TRBC (QN) Indirect Coombs Test (QL)
    TLC (QN)  
  Factor VIII (QN)    
  Fibrinogen (QN)    
  PTTK (QN)    
  Pyogenic Culture (QN)    
 
EQUIPMENT/REAGENTS WHICH NEED IN-HOUSE CHECKS (IQC)
 
  • ELISA Reader/Kits
  • Blood grouping equipment/Reagents
  • Cross Matching cards/Antibody screening
  • Hematology analyzer/Reagents
  • pH meter
  • Rapid cards/RPR/MP
  • Coagulation analyzer
 
Internal Quality Control for Quantitative Tests
 
  •   Implementing QC program for Quantitative tests operates on the following steps:
  •  Select quality controls.
  •  Collect at least 20 control values over a period of 20- 30 days for each level of control.
  •  Perform statistical analysis.
  •  Develop Levey-Jennings chart.
  •  Monitor control values using the Levey-Jennings chart and/or Westgard rules.
  •  Take immediate corrective action, if needed.
  •  Record actions taken.
 
SELECTING CONTROL MATERIALS/CALIBRATORS
 
A calibrator has a known concentration of the substance (analyte) being measured. Calibrator is used to adjust instrument, kit, test system in order to standardize the assay. Calibrator is not a control.
A control has a known range of the analyte. Usually 2 or 3  levels of controls are used based on the number of tests run daily for a particular analyte. The controls should be run along with patient sample and are used to validate day to day reliability of the test system.
 
Characteristics of Control materials
 
  •  Different values covering all Medical decision points
  •  Similar to the test specimen (matrix)
  •  Available in large quantity
  •  Stored in small aliquots
  •  Ideally, should last for at least 1 year
 
Managing Control materials
 
  •  Sufficient material from same lot number/ generation number or serum pool for one year’s testing
  •  May be frozen, freeze dried, or chemically preserved
  •  Requires very accurate reconstitution if this step is necessary
  •  Always store as recommended by the manufacturer
 
Sources of QC samples
 
  •  Appropriate diagnostic sample Obtained from:
  •  Another Laboratory
  •  EQA provider
  • Commercial product

 

Types of Control materials ASSAYED:
 
Mean calculated by the manufacturer

Must verify in the laboratory
 
UNASSAYED:
 
Less expensive

Must perform data analysis
 
“HOMEMADE” OR “INHOUSE”:
 
Pooled sera collected in the laboratory

Characterized

Preserved in small quantities for daily use
 
ANALYSIS OF QC RESULTS
 
Analytical tools are needed for the analysis of the QC data. Basic Statistical skills are required for the  urpose and form an important skill for the laboratory personnel. Levy Jennings’s (LJ) chart should be used to plot daily QC values. It indicates the changes in trends and shifts of the laboratory performance.

Westgard rules should be used to interpret daily QC values. The level of QC applied in the laboratory varies according to the number of specimens analyzed per day. A data set of at least 20 points should be obtained over a 30 day period. These data points are used to calculate mean, standard deviation, coefficient of variation; determine target ranges. It is made sure that all the procedural variation is represented including different operators, different times of the day. Variability in the data is determined to establish the acceptable range.
 
MEASUREMENT OF VARIABILITY –
 
A certain amount of variability will naturally occur when a control is tested repeatedly. Variability is  ffected by operator technique, environmental conditions, and the performance characteristics of the assay method. The goal is to differentiate between variability due to chance from that due to error. The terms used to describe variability are: Range, Variance, Standard Deviation, Coefficient of Variation.
 
MEASUREMENT OF CENTRAL TENDENCY –
 
  • Data are frequently distributed about a central value or a central location.  The measures are –
  •  MEDIAN - The value at the center(midpoint) of the observations
  • MODE – The value which occurs with the greatest frequency
  • MEAN – The calculated average of the values
 
CALCULATION OF MEAN –
 
done using the standard formula keeping out of calculation the outliers (values which fall outside the manufactures supplied control range).
 
MEAN FOR ELISA TEST
 
The calculation of mean is especially useful for ELISA test used in blood banks. The optical density (OD) values and cut  off (CO) for controls for each assay run is measured. The ratio of OD to CO (OD/CO) is calculated and considered as a single data point. This ratio standardizes the data and is used to calculate the laboratory mean.
 
where X = OD/CO (OD- optical density; CO - cut off for control)
 
  • NORMAL DISTRIBUTION; X AXIS – RATIO OF OD/CO;
  • YAXIS – FREQUENCY
  • MEAN FOR HEMATOLOGY PARAMETERS
 
where X = value of test eg. Hb, Platelet count etc ACCURACY AND PRECISION
 
  • The degree of fluctuation in the measurement is indicative of the “precision” of the assay.
  • The closeness of measurements to the true value is indicative of the “accuracy” of the assay.
  • Quality control is used to monitor both the precision and the  accuracy of the assay in order to provide reliable results.
RANGE
 
Refers to the difference or spread between the highest and lowest observations. It is the simplest measure of dispersion. It makes no assumption about the sahpe of he distribution or the central tendency of the data.
 
VARIANCE (S2)
 
Is a measure of variability about the mean. It is calculated as the average squared deviation from the mean.

S2 = ?(X-X¯) 2 = mg2/dl2

N-1
 
DEGREES OF FREEDOM
 
  • Represents the number of independent data points that are contained in a data set.
  • The mean is calculated first, so the variance calculation has lost one degree of freedom (n-1).
CALCULATION OF STANDARD DEVIATION
 

The standard deviation is the square root of the variance; it is the square root of the average squared deviation from the mean. SD is commonly used (rather that the variance) since it has the same units as the mean and the original observations. SD is the principle calculation used in the laboratory to measure dispersion of a group of values around a mean. Laboratories use the +/- 2SD criteria for the limits of the acceptable range for a test meaning that when the QC measurement falls within that range, there is 95.5% confidence that the measurement is correct. Only 4.5% of the time will a value fall outside of that range due to chance; more likely it will be due to error. S = square root of (X-X¯) 2 = mg/dl

N-1

S = square root of Variance

CALCULATION OF COEFFICIENT OF VARIATION
The CV is the standard deviation expressed as a percentage  of the mean. Ideally it should be less than 5%.
CV = SD/mean X 100
 
MONITORING OF QC DATA
Levy Jennings’s (LJ) chart should be used to plot daily QC  values for each run and make decisions  regarding acceptability of run. Monitoring should be done to

evaluate the precision and accuracy of repeated measurements. Charts needs to be reviewed at regular intervals to take necessary action and

documented.
 
LEVEY-JENNINGS CHART

It is a graphical method for displaying control results and evaluating whether a procedure is in-control or out-of-control. The control values are plotted versus time.

Lines are drawn from point to point to accent any trends, shifts or random excursions.

 
An example of a Levey-Jennings chart with upper and lower limits of one and two times the standard deviation.
The control values in a L-J chart should be ideally clustered about the mean (+/- 2SD) with little variation in the upward or downward direction.
 
IMPRECISION – large amount of scatter about the mean  usually cause by error in the technique
 
INACCURACY – may be seen as a trend or a shift, usually caused by change in the testing process.
 
RANDOM ERROR - No pattern. Usually poor technique, malfunctioning equipment.
 
WESTGARDRULES

Multirule QC uses a combination of decision criteria, or control rules, to decide whether an analytical run s in-control or outof- control. The well-known Westgard

multirole QC procedure   uses 5 different control rules to judge the acceptability of an analytical run. By comparison, a single-rule QC procedure uses a single
criterion or single set of control limits, such as a Levey-Jennings chart with control limits set as either the mean plus or minus 2 standard deviations (2s) or the
mean plus or minus 3s. “Westgard rules” are generally used with 2 or 4 control measurements per run, which means they are appropriate when two different
control materials are measured 1 or 2 times per material, which is the case in many chemistry applications. Some alternative control rules are more suitable

when three control materials are analyzed, which is common for applications in hematology, coagulation, and immunoassays.

 

13s Refers to a control rule that is commonly used with a Levey-Jennings chart when the control limits are set as the mean plus 3s and the mean minus 3s. A

run is rejected when a single control measurement exceeds the mean plus 3s or the mean minus 3s control limit.

 

12s Refers to the control rule that is commonly used with a Levey-Jennings chart when the control limits are set as the mean plus/minus 2s. In the original

Westgard multirule QC procedure, this rule is used as a warning rule to trigger careful inspection of the control data by the following rejection rules.

 

22s Reject when 2 consecutive control measurements exceed the same mean plus 2s or the same mean minus 2s control limit.

 

R4s Reject when 1 control measurement in a group exceeds the mean plus 2s and another exceeds the mean minus 2s.

 

41s Reject when 4 consecutive control measurements exceed the same mean plus 1s or the same mean minus 1s control limit.

 

10x Reject when 10 consecutive control measurements fall on one side of the mean.

 

When a rule is violated; in case of WARNING RULE use other rules to inspect the control points. In case of REJECTION RULE (Out of control) immediately stop testing – identify and correct the problem – repeat testing on patient samples and controls – do not report patient results until problem is solved and controls indicate proper performance.


For solving out of control problems there should be definite policies and procedures for remedial action; troubleshooting guides and alternatives to run rejection

 

               Internal Quality Control for Qualitative Tests

 

Qualitative test (rapid card) for transfusion transmitted disease with built in controls are adequate  rovided the kit has been qualified with at least one positive and  one negative control from external sources (can  e in-house also). These shall be validated by two qualified personnel and documented. Qualitative test in blood group serology is carried out daily and documented as follows:


 


Sr. No Reagents Performance Frequency Of Testing
1. Anti Human Serum            Appearance, Reactivity And Specificity         

Daily

2. Blood Grouping Serum Appearance,Specificity,Avidity,Reactivity,Potency

Daily

3. Antibody Screening And Appearance , ReactivityAnd Specificity  

Lot

  Reverse Grouping Cells    
4. Syphilis (Rapid Card)    Positive And Negative Control (External)    

Each TestN

5. Normal Salinee Appearance, Nacl Content, Ph, Haemolysisl   

Daily

 
Daily Equipment/Reagents
Sr. No Equipment Performance Frequency Of Testing
1. Temperature Recorder        Compare against thermometer      

Daily

2. Refrigerator Compare against thermometer     

Daily

3. Gen Centrifuge        RPM

Standardize speed before initial use

4. Blood bag     Centrifuge Temperature RPM Every        

Fortnight

5. Water bath   Observe temp

Daily

6. Auto-Clave            Observe temp

Daily

7. Cell counter Calibration Reference sample

Daily

8. Balance Analytical control.  
  Weights 5mg-100g 6 Monthly  
9. Platelet Agitator Frequency of agitation

Monthly

10. Laminar Flow hood Air pressure, clean

Monthly

  Gen/Electronic thermometer Observe temperature

Daily

 
IMPLEMENTATION OF INTERNAL QUALITY CONTROL PROGRAM
 
  • Establish written policies and procedures
 
  •  Assign responsibility for monitoring and reviewing
 
  • Train staff
 
  • Obtain control materials
 
  • Collect data
 
  • Set target values (mean, SD)
 
  • Establish and implement troubleshooting and corrective action protocols
 
  •  Establish and maintain system for documentation
 

 

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Dr. Rohit Jain
MD
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