Determination of acceptable levels of haemolysis when accepting blood samples for trace mineral analysis

Bronwyn Claudia Cloete

The effect of haemolysis on the quality of results in the Biochemistry laboratory

It’s all about Quality“A process is a collection of activities

that converts inputs into outputsor results” - Gryna, Chua & DeFeo (2007:195)

A collection of processes is asystem

ISO 9001:2008 advocates the Process Approach

Coffee Making Process:

Specific requirement: Morning CuppaSpecific inputsSpecific stepsEvaluate outcomeDetermine improvement needsImprove

QUALITY IS APPLICABLE TO EVERYTHING

ISO 9001:2008What the customer wants is what the

customer getsProcess Approach:

Inputs Outputs

Based on customer requirementsWithout customer, there will be no need for

QualityOverview of ISO 9001:2008

PROCESS

SANS 17025: 2005

Identical implementation to ISO/IEC 17025:2005

Overview of ISO 17025:2005ISO/IEC 17025:2005 is based on ISO 9001No compliance to ISO/IEC 17025 without

compliance to ISO 9001New standard due soon

Improvement = Object of StudyQuality Improvement needs to add practical

value

Research Design: Primarily scientific with elements of social science methodology

Structure of research design: Action research in the positivistic paradigm

Experimentation for data collectionProtocol analysis and Observation data

collectionAu fait with study environment

Indentify Customer Requirements

Research Objective: Determine exact values of acceptable levels of haemolysis when accepting blood samples for trace mineral analysis.

Primary Research Question: What is the maximum haemolysis level acceptable, as measured in terms of optical density using a spectrophotometer at 540 nm wavelength, in order to accept samples for Trace mineral analysis?

Literature Review Saibaba (1998: online) states presence of substance in a sample having effect which changes correct value of the

result must be corrected to ensure quality (Saibaba, 1998: online)  Thomas (2010: online) contends that haemolysis is an important interference factor (Thomas, 2010: online)  Guder. (1986: online) asserts: “Haemolysis is defined as the breakdown of red blood cells and the release of

haemoglobin and intracellular contents into the plasma and Plasma concentrations exceeding 300mg/L, results in the haemolysis of red blood cells being observable to the naked eye. (Gruder, 1986: online)

 Lippi, (2009: online) states major worldwide concern for all clinical laboratories is in vitro haemolysis affecting test results and seriously impacts on patient care and the laboratory’s reputation. (Lippi, 2009: online)

 Ong, Chan, Lim (2009: online) conducted studies finding that a cost saving occurred with a reduction in sample hemolysis (Ong et al., 2009: online)

 Henry, Cannon, and Winkelman, asserts that Spectrophotomic methods can be used to read hemoglobin levels. (Henry et al., 1974 (6))

 Spectrophotometers are standard research tools, used in chemistry laboratories, utilizing the relationship absorption of light and colour as principle for the way it works. (Hoydt, n.d.: online)

 To achieve impact from research, the research environment is examined from holistic perspective. Ruiz-Marrero (2009: online) contends the holistic view, is an “interdisciplinary vision conceiving every natural system as an integrated whole, which cannot be understood if broken down into its constituent components” (Ruiz-Marrero, 2009: online)

 In additional to holistic consideration the research environment Jiju Mike, Andreas, (1998: 169 - 176) contends the use of statistical quality control techniques is an essential part of the search for effective quality control and can lead to quality improvement id applied correctly. (Jiju et al, 1998: 169 - 176)

 According to The Quality Assurance Project,(QAP), (n.d.: online) quality improvement involves applying methods most appropriate in order to close the gap between expected levels of quality and current levels of quality.

 Gate to Quality (n.d.: online) asserts that “Any tool or technique that can be used for improving the process/product quality, help in analyzing the current situation, help in gathering information or help in bringing small or big change (towards improvement) in the organization can be called a Quality tool or technique.” (Gate to Quality, n.d.: online)

Identify and Examine ProcessQuality engineer’s toolbox7 Basic Quality Tools (B7)

PDCA cycleAdditional: 7 New Quality Tools for project

management (N7)

Plan Phase : Background ResearchBased on research objective the following were

examined in the research environment:

QMSEquipment resourcesReagents and methodsStaff componentOther Process Inputs: Samples

Ishikawa Root Cause Analysis

Ishikawa Diagram

Sample pie chart

Existing Process MapQuality Management SystemAll systems in Biochemistry

Reception: Samples arrive at LabSample information captured

Sample Analysis:Verifiable analytic method according to SOP.

Controlled conditionsUse of Controls and Standards

Critical Suppliers

Quality Management Documents and Records

Satisfied Service Customer

Technologist reviews resultIssues it for release from Biochem

Validation: Checks performed to see if SOP followedControls and Standards in spec

Veterinarian reviewsCompiles with results from other labs

Issues report

Samples delivered to Biochemistry Section

Sample Reception at BiochemistrySamples information recorded.

Biochemistry lab number assigned.Test Allocation.

Samples stored under ideal conditions until testing

Record keeping

YES

NO: Corrective action involves

redo

PDCA Cycle

PLAN, DO, CHECK and ACTCycles for continuous improvementAlso known as PLAN, DO, STUDY and ACT

(PDSA)

Dr William Edwards Deming1900 - 1993

Statistician, professor and authorConsidered the Father of QualityRenowned for work conducted post

World War II and credited for the “Japanese Industrial Miracle”

Along with Juran, launched Total Quality Management

“Learning is not compulsory ...neither is survival “ – Dr. William E. Deming

Do Phase : Conduct an Experiment

Sample group of ten ewes, nonpurposively sampled.

Four samples of blood collected in red top serum tubes from each ewe on fortnightly basis.

One tube from each animal centrifuged immediately, serum removed from clot.

Artificial manipulation remaining three tubes, blood stored in 25oC incubator and every third day a tube was centrifuged and serum removed.

Samples stored in 4oC fridge until analysis.

Do Phase : Conduct an Experiment

Procedure repeated for ten replicates over a twenty week period.

Thus: Ten sets of data collected for each animal in the sample group.

All samples were spectrophotometrically measured.Each data set contained measurements of the

following trace elements: Cu, Zn, Ca, Mg, Phos and Fe.

Measurements were taken of Fresh samples, Day three, Day six and Day nine samples.

Check Phase : Analyse the dataRaw data analysed with statistical quality

tools

SPC : Control charts, ANOVA hypothesis testing and regression and correlation

Check sheets, scatter diagrams

CUSTOMISE THE BOUQUET OF QUALITY TOOLS FOR YOUR OWN PURPOSE

Average Spectrophotometric Measurements

103 104 114 117 119 120 123 124 129 1410

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Average Haemolysis Readings

Fresh Day 3 Day 6 Day 9

Fresh Day 3 Day 6 Day 90

0.2

0.4

0.6

0.8

1

1.2

Average Haemo-lysis Readings

Series1

Copper (Cu)

Mean copper mineral levels were significantly out of recommended normal range, as expected due to known environmental factor of soil copper levels in the Stellenbosch area being traditionally low. Copper levels were elevated with the upward progression of haemolysis in samples. This elevation was detrimental to result quality output even though it was within recommended normal range.

Zinc (Zn)

Mean zinc mineral levels stayed within recommended normal range, however a trend was observed whereby the mean zinc levels were elevated due the upward progression of haemolysis levels. Thus it can be said that uncontrolled haemolysis in samples would have a detrimental effect to result quality output.

Calcium (Ca)

Mean calcium trace mineral levels were initially found to be within the recommended normal range, however with the upward progression of haemolysis, the mean calcium levels were found to significantly decrease. Thus uncontrolled haemolysis in samples would have a detrimental effect to result quality output. Group 9 mean calcium levels were out of recommended normal range.

Manganese (Mg)

Mean magnesium trace mineral levels stayed within recommended normal range, however an upward trend was detected in relation to the upward progression of haemolysis.

Phosphorous (Phos)

The mean phosphorous trace mineral level s of the fresh group was found to be within recommended normal range, however with the upward progression of haemolysis the mean trace mineral value steadily moved upwards and significantly out of recommended normal range.

Iron (Fe)

The mean iron trace mineral level of the fresh group was only slightly out of recommended normal range. It was determined that this could also be as a result of environmental factor impacting on research, however the significant observation to be made during interpretation, is considered to be the steady and marked upward trend of mean iron levels with the upward progression of haemolysis occurrence in samples. Mean iron levels of groups Day 3, 6 and 9 are considered to be significantly out of recommended normal range.

ANOVA and Questionnaire Analysis

With the exception of the trace element Zn an ANOVA f test conducted tested the hypothesis that the haemolysis effect on the results of all the other trace element was significantly different.

Protocol analysis and observation studies were conducted. Statistical analysis of feedback from staff members provided a scale for a colour chart.

Quality Improvement AreasSeptember 22,

2010

Page 1

Chart Type

Survey Sample - 8 Categories

Operational Systems in Biochemistry Environment

100

90

80

70

60

50

40

30

20

10

LegendPre-analytical

FactorsEquipment

Factors

Process Factors External Factors

Support Requirements Defined: Documentation and Records

Process Structure: Logical, sequential,

comprehensive

Process Requirements Defined: Documentation and Records

Corrective Action/

Preventative Measure in

system

Control Measures in system

Traceabilty and adequate Identification of samples in system

Calibration in system

Effectivity

Notes:

This chart illustrates the results of protocol analysis on the operational environment of biochemistry laboratory in relation to the various systems which comprise of it namely:

· Pre-analytical Systems· Core Process Systems· Equipment Factor Systems· External Systems input

A graphic representation is given, enabling the user to identify the strengths as well as the weakesses in each of the pre-defined systems

Act Phase : ResultsQMS in Biochemistry section at WC PVL is good.Inadequate samples have detrimental effect on result quality.Haemolysis influences trace mineral levels of samples.Invalidates normal reference values.A linear relationship between haemolysis levels and all trace

mineral levels researched.A linear positive relationship exist between time before

centrifugation takes place and haemolysis level.Extent of the relationship is different from trace elements to

trace element.Haemolytic impact found to be progressive over time in all

cases.

Conclusion : Recommendations

Samples for trace element analysis must be screened for suitability for analysis.

A colour chart to be used as a practical measure in order to screen samples received for trace mineral analysis at WCPVL. 

If uncertainty prevails over acceptability of a particular sample, a spectrophotometric test may be done to establish that the sample does not exceed the maximum acceptance level of 0.377nm.

Colour Chart

Henry, Cannon, and Winkelman, assert that spectrophotomic methods can be used to read hemoglobin levels. (Henry et al., 1974:6)

Modified Process MapReception: Samples arrive at Lab

Sample information captured

Samples delivered to Biochemistry Section

Sample Reception at BiochemistrySamples information recorded.Biochem lab number assigned.

Test Allocation.Samples stored under ideal

conditions until testing

Veterinarian reviewsCompiles with results from other labs

Issues report

Technologist reviews result

Issues it for release from Biochem

Validation: Checks performed to see if SOP

Followed. Controls and Standards in spec

Sample Analysis:Verifiable analytic method

according to SOP.Controlled conditions

Use of Controls and Standards

Quality Management SystemAll systems in Biochemistry

Critical Suppliers

Record keeping

Satisfied Service Customer

Quality Management Documents and Records

Additional Step:Screening Test

NO: Corrective action involves

redo

YES