Mean Reticulated Haemoglobin (Hb) Content (RetHe) Analysis of Renal Patients
Condition: End-Stage Renal Failure; Functional Iron Deficiency
MeSH terms: Kidney Failure, Chronic; Anemia, Iron-Deficiency; Iron Deficiencies
Study type: Observational

Summary: 
      Anaemia is a common complication of Chronic Kidney Disease (CKD) the management of which has
      been aided by the use of synthetic recombinant human erythropoietin therapy (r-HuEPO). This
      red cell stimulating agent creates the further complication of Functional Iron Deficiency
      (FID) where, despite normal iron stores, patients fail to respond to therapy as they do not
      possess enough available iron to meet the demand of increased red cell production. Effective
      response to r-HuEPO therapy depends on an appropriate monitoring of 'available' iron levels.

      Previous research into the clinical utility of testing for reticulated haemoglobin
      concentrations (Ret He) instead of Serum Ferritin and Transferrin Saturation analysis has
      indicated an advantage as an iron deficient prognostic marker however, further knowledge is
      required on the use of this new laboratory test (RetHe) to predict Functional Iron Deficiency
      (FID) level and to study it's relationship with responses to therapy.

      This proposed study aims to estimate a local working Normal (non deficient) and Iron
      Deficient Reticulated Haemoglobin Content (RET He) reference range from surplus anonamous
      samples. Routine monthly blood samples from Pre Dialysis and Haemodialysis patients will be
      used to evaluate the sensitivity and specificity of the RET He test compared to current
      laboratory tests and investigate its predictive ability for Functional Iron Deficiency in
      these patients.

      Studying , measuring and statistically analysing the change in the RET He parameters in
      Haemodialysis and Pre Dialysis patients over 3 months will look for evidence of a direct
      relationship between RET He values and the patients response to therapy. The data will be
      used to provide a predictive picture of what levels of RET He indicate Functional Iron
      Deficiency.

      The introduction of this test (RetHe) may provide clinicians with a one sample/one test
      control over iron therapies and ensure the patient gets the most benefit from erythropoietin
      therapy.
    

Detailed description: 
      Reference Range Testing for new test RET-He will be performed on excess blood available on
      randomly selected anonamised samples obtained from the daily workload of the laboratory once
      all the clinically requested analysis has been completed.

      100 samples (50 men / 50 women)determined to have normal parameters(as defined by Dacie et
      al, 10th Edition) and 100 samples (50 men / 50 women)determined to be iron deficient (as
      defined by Dacie et al, 10th Edition)will be processed for new test parameter (RetHe) under a
      coded number to ensure no identifying data is recorded. The samples will be archived and and
      held within the lab for 24 hours prior to routine disposal in accordance with NHS Fife Waste
      Management Policy.The data will be analysed using a paired t test to determine significant
      differences and reference range inferred inferred.

      Evaluation of sensitivity / specificity of new Ret He test in determinimg iron deficiency and
      functional iron deficiency and it's response to terapy will be tested using Haemodialysis and
      Pre-Dialysis Patient Samples (~180 Haemodialysis Patients and ~250 Pre Dialysis Patients)that
      are obtained during their normal renal unit / clinic appointments.

      All consenting NHS Fife Haemodialysis /Pre Dialysis patients will be allocated a 'Subject
      Identification Number' as part of this study (which will be noted at the top of their Consent
      Form). This number will be used by the Student Investigator to generate a confidential Coding
      Key which will be kept separate from data information and secure uner an NHS password
      protected computer. This will ensure care of personal information and will be used
      through-out the study by the student investigator to record all generated data.

      Patients will be grouped into categories such as Dialysis / Pre Dialysis on a coded SPSS
      (statistical software) file.

      All consenting patients will attend their routine scheduled Renal Clinic appointments as
      normal (no additional appointments are necessary for this pilot study)and their response to
      therapy will be observed using the new RetHe test parameter compared to existing laboratory
      tests utlised (FBC,Ferritin and Transferrin Saturation Levels)

      Haemodialysis Patients - June 2010, July 2010*, August 2010

      (* NB: In July 2010 Haemodialysis patients are scheduled to have their Ferritin and
      Transferrin Saturation Levels checked and therfore no additional sample will be required)

        1. Routine venesection of EDTA (purple topped) sample for Full Blood Count (FBC) Test

        2. Routine venesection of Clotted (yellow topped) sample for Biochemistry Analysis

        3. Additional venesection of extra yellow topped sample for non routine Ferritin
           Test(*usually scheduled every 3 months- routine date due: July 2010)

        4. Renal Unit Staff label samples and complete request form:

           Haematology section ticked for: FBC (as normal routine) , Retics (to allow addition of
           new Ret He test to FBC analysis) and Ferritin (additional request in June and August)

           Biochemistry section ticked for : All routinely requested Biochemistry tests (such as
           Urea and Electrolytes) plus additional request for Transferrin Saturation Levels (in
           June and August)

        5. Sample and Requests sent to Fife Area Lab for Analysis

           The project will be carried out within NHS Fife Area Laboratory Haematology Department
           and will involve the use of NHS premises, facilities and staff.

        6. Request checked at Laboratory Central Reception (sample and request form labelled with
           unique positive barcode identification)

        7. Request Forms sent to central request entry and samples delivered to appropriate
           laboratories

        8. FBC samples checked prior to analysis to check request details entered into Laboratory
           Host Computer System

        9. FBC sample loaded onto Sysmex XE2100 Haematology Analyser for testing

       10. Bidirectional Computer Link between Laboratory Host Computer System and Analyser ensures
           sample tested for FBC and additionally requested RETIC (reticulocyte analysis)

       11. Sysmex XE2100 automatically analyses sample for Red Blood Cell Indices and additionally
           uses fluorescent polymethine dye to stain for RNA/DNA content of reticulated cells
           (early red blood cell precursors). The analyser uses inbuilt Flow Cytometry and measures
           forward scatter to separate cells by size (this is reported as RBC-Y and RET-Y)

       12. Sysmex XE2100 RETIC Master software (recently upgraded packaged loaded onto NHS Fife
           Analysers)uses an algorithm (RET-He = 5.5569e 0.001RET-Y) to convert RET-Y into a
           reportable mean reticulocyte haemoglobin content (RET He) which is expressed in
           picograms.

       13. The sample once analysed is removed from the analyser and the results are either
           autovalidated and archived (if results are within normal range) or held for validation
           by a NHS Fife qualified Biomedical Scientist.

       14. The routinely requested FBC results are available for clinicians within same timescale
           as normal (no delay due to additional RetHe analysis)

       15. The sample is archived under their unique identifying barcode numbers and held within
           the lab for 24 hours prior to routine disposal in accordance with NHS Fife Waste
           Management Policy.

       16. The new RetHe value will be held on Sysmex XE2100 analyser (not activated as a
           reportable test to NHS Fife Biomedical Scientists or clinical staff.)

       17. The Student Investigator will (on same day as analysis) retrospectively generate a Renal
           Unit worklist from the Laboratory Host Computer System which will indicate: how many
           samples received, barcode numbers, names of patients.

       18. The Student Investigator will (on same day as analysis) retrospectively use the
           generated worklist to search the Sysmex XE2100 analysers for Ret-He test results and the
           host computer system for results of Ferritin and Transferrin Saturation Levels.

       19. The barcode number of the patient and their test results will initially be recorded in
           the student investigators laboratory handbook (the generated worklist will be discarded
           into NHS Fife Confidential Waste)

       20. The student investigator will record the test values into a coded SPSS (statistical
           software) file for appropriate analysis.

       21. The investigator will retrospectively match the test data with therapy regime data
           provided by the Renal Unit (monthly meetings)

      Pre Dialysis Patients: June 2010, July 2010, August 2010

      (NB: Pre Dialysis Patients are tested 3 monthly so over period of study should get a baseline
      sample from each consenting patient. Only those patients who are considered iron replete and
      are placed on 5 week course of iron therpay will be further tested at their follow-up
      appointments in order to measure response to therapy)

        1. Routine venesection of EDTA (purple topped) sample for Full Blood Count (FBC) Test,
           clotted (yellow topped) for Ferritin and clotted (yellow topped)sample for Transferrin
           Saturation Levels

        2. Renal Unit Staff label samples and complete request form:

           Haematology section ticked for: FBC (as normal routine) , Retics (to allow addition of
           new Ret He test to FBC analysis) and Ferritin (routine request)

           Biochemistry section ticked for : All routinely requested Biochemistry tests (such as
           Urea and Electrolytes) plus routine request for Transferrin Saturation Levels

        3. Sample and Requests sent to Fife Area Lab for Analysis

      The project will be carried out within NHS Fife Area Laboratory Haematology Department and
      will involve the use of NHS premises, facilities and staff (same process as points 6 - 21
      above)
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Renal Patients requested to participate in study (consent required)
      
Keywords: Reticulated Haemoglobin Equivalent; Functional Iron Deficiency; Response to therapy; Iron deficiency