Quality Management in Hematology Laboratory.

Quality Management in Hematology Laboratory.
By Heidi Hanes

This project has been funded in whole or in part with Federal funds from the Division of AIDS (DAIDS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services Contract No. HHSN C Project Title: Patient Safety Monitoring in International Laboratories.

Objectives At the end of this presentation should be able to:
Recognize the four components of Quality Assurance. Recognize how to troubleshoot problems. Develop own policy for all aspect of quality assurance. Understand what is necessary to undertake a program of Quality Management in Hematology Laboratory.

Quality Management includes
Standardization Pre Analytical Control Post Analytical Control Analytical Control Internal Quality Control External Quality Assessment Quality Management includes the following:

So let’s begin our journey.
Quality assurance is essential in hematology to ensure that laboratory tests are carried out reliably. Quality assurance is concerned with all steps of laboratory practice. The process from specimen collection included in the pre-analytical portion to transmission of the report to the clinician which include the post analytical control. And the analytical process inbetween.

Standardization Collaboration between groups.
Standard Operating Procedures Problem solving for unsatisfactory performance Test selection The First– standardization has occurred over the years with collaboration between such groups as WHO-World Health Organization- and International Council for Standardization in Hematology. They have promoted the standardization of methods and the use of reference material standards to ensure correct resulting. An example of a standard used in the hematology lab is the standard used for calibrating hemoglobin. All calibration material is tested against it. The creation of SOP help to maintain quality of performance by providing a stable pattern of function and avoiding unauthorized individual variation. Ensures consistent quality of work no matter what skill level from new to seasoned staff This can involve a number of procedures such as calibration of the instrument, changing of parts/tubing. Depending on the instrument it may be necessary to rely on technical service by the manufacturer. But regular preventive maintenance of any instrument will reduce the incidence of problems. Minimizing the risk of breakdowns. And finally the actual choosing of what test will be run. The test performed should provide either quantitative or qualitative information relevant to specific patients or to the general population. Choice an instrument that has the test you really need. In our Resource section on psmile.org website there are several documents to help you in the selection process. There are multiple checklist you can use to get through this process to help pick the right instrument for your needs.

Pre-Analytical Control
We will start with what is included in the pre-analytical control section.

Understanding functionality of your instrument:
principles of operation startup or daily checks shutdown procedure normal sights and sounds of the instrument familiarize staff to troubleshooting manual Non-technical people will say that the instruments can run themselves now you don’t need to know anything just put the blood on the instrument and push a button and then you get the results. This is so untrue. It is very important for all staff to understand what happens to that blood is sampled by the instruments. When they get the results which is involved in the Post analytical they need to decide if the results are correct. If there are problems then you need to be able to troubleshoot where the inaccurate result came from. Is it from the sampling valve? Are the pressures and vacuums correct to move the sample about the tubing. Is there a problem with the reagents? All of these questions can be answered if your staff to have a good understanding of the working of the instrument. Performing startup and daily checks is another important area. The results can also tell you if your instrument is functioning correctly. Then at the end of work day the shutdown. As we all know blood is very sticky mainly caused by the protein. This protein will coat all the tubing and parts of your instrument so that shutdown is important because the reagent will clean those surfaces. Next knowing what are the normal sounds and sights of your instrument. When you walk up to it you should be looking to make sure you don’t see any leakage. Same with any unusual sounds. (When it comes to instruments/cars I can detect the smallest difference in them through sound. It would drive my husband crazy when I got into his car. I would say “What is that noise” He would say there is no noise you just are hearing things. The technical hot line people would be amazed that I could detect a bad fan way before it finally broke.) Part of staff training with a new instrument is familiarizing them to the troubleshooting manual.

Hematology Subsystems
Consist of 3 subsystems. Electronic Fluidic Pneumatics (pressure and vacuums) Hydraulics (liquids) Reagent On most hematology instruments they are broken into three subsystems Electronics monitor all of the functions of the instrument such as lamp voltage on the hemoglobin lamp. It also includes all of the computer boards that are involved with taking the analyzed data and outputting the actual results The Fluidic subsystem include both pneumatics that are the pressure and vacuums that move the fluids around the instrument and the actual hydraulics or liquids. It includes tubing, pinch valves, pumps and solenoids. The older instrument used mercury in the vacuum system. Periodically it would have to be changed because it got dirty. This was back when no one knew that it was hazardous to come in contact with it. (We would play with it make the drops come together. Has anyone seen any of the new Terminator films. It makes me think of the new terminators.) The other is the actual reagent. If you don’t have a drain for the waste but instead have it connected to a box this is included.

Proper Specimen Collection
Analyze only specimens that were properly collected and stored. Correct Tube Correct amount of specimen in tube Proper mixing Cleanliness of puncture area Storage Another aspect of Pre-analytical is Proper Specimen Collection. Each manufacturer have specific tube type that can be used as the anticoagulant. Example for Coulter only lavenders and blue tops or hematology finger sticks can be used. If you use a tube containing heparin it will react with the reagents producing precipitates in the instrument. Something that you don’t want because it will take you forever to get it all out of the tubing. Also if is important the order of tubes that are drawn. You know that you should never draw a blue top for coag after a tube that had heparin in it. If too little blood the sample can be diluted out given false low results. If too much then there is a possibility of clotting of the specimen. For sites that are participating in Mother/Child research includes pediatrics draws. These can be either finger/heel sticks or venous punctures. All of the tubes require specific amount of blood to give accurate results. For laboratory that get blood draws from clinics out in the field could mean that the blood may sit for a length of time. So once they get to the lab if not mixed enough than can have inaccurate results. On the other hand if mixed too hard could cause cell damage leading to inaccurate counts. Cleanliness is important especially with micro collections or finger sticks. Don’t want to introduce extra cells or material such as epithelial cells. Also in finger sticks the amount of pressure used can change the make up of the blood such as introduction of tissue fluid or hemolysis Storage – in a perfect world you are able to get that blood to the lab and run within a couple of hours. We all know that is not the case in many of the clinics that are distant from the main lab. The samples have to be carefully maintained. Again this can vary between instrument manufacturers. Always check what is recommended by your instrument manufacturer. They usually have the storage recommendation for temperature and length of time between draw and running of samples. If your site can’t do these recommendation then you should validate any different storage conditions to ensure accurate results. (One study we did at my laboratory for our coagulation testing. We wanted to increase the time between collection and running of the test from manufacturer’s recommendation. We tested 10 normal, 10 warfarin/coumadin and 10 heparin bloods. We drew 2 blue tops for each group. Ran all of them for the initial result then stored one capped at room temperature and the other capped and refrigerated. We then reran them at specific intervals to see if there was any change. At the end of our testing we found that the normal and coumadin samples could be kept at room temperatures for 36 hours without any affect to the PT result. The heparin samples were only good for maximum of 4 hours unrefrigerated for APTT. Both normal and Coumadin samples showed acceptable results up to 6 hours unrefrigerated for the APTT.) So now we will look at a couple of examples for troubleshooting pre-analytical problems.

Exercise 1

Pre-Analytical Example 1
21-May-2015 13:45 WBC 0.05 x 10^3/L PASS RBC 0.00 x 10^6/L PASS Hgb 0.00 x g/dl PASS Plt 10.0 x 10^3/U FAILED Example 1 - Here we have a background printout after start-up. As you can see the Platelet background failed. First would look at your troubleshooting guide. What would you think most likely first step would be? Perform another background count.

Repeat Background 21-May-2015 14:00 WBC 0.05 x 10^3/L PASS
RBC 0.00 x 10^6/L PASS Hgb 0.00 x g/dl PASS Plt 15.0 x 10^3/U FAILED Here is our rerun and still platelets are out even more. Once again would check your troubleshooting SOP.

Corrective Action Check when reagent(s) last changed.
Change the most recently added reagent. If none recently added change the diluent. Rerun start-up Here are some possible corrective actions to perform Why is this important? If there is a new reagent it could be the one causing the high count. Next step change the reagent. If none has been change recently the reagent that could cause the problem is the diluent since it is the highest portion of this background sample. Once changed then would re-do the startup.

Repeat Background 21-May-2015 14:30 WBC 0.05 x 10^3/L PASS
RBC 0.00 x 10^6/L PASS Hgb 0.00 x g/dl PASS Plt 2.0 x 10^3/U PASS So for our example background is in and we can continue with our daily maintenance. But just like any problem you need to document the corrective action and try to figure out possible causes of it.

Causes for high background
The uptake tubing got contaminated. Reagent agitated before connecting to instrument. Dried reagent spills or leaks. So let’s look at possible causes to the high background. Part of the procedure for changing anything on the instrument including reagent should be documented and include the personnel initial so you can ask them questions. Contamination of the reagent. If a reagent had been changed recently ask person that changed the reagent to demonstrate what they did. Might have laid the tubing on the floor or touched it with contaminated hands. Tech shows rolling the box over to the instrument due to it large shape. This would introduce bubbles that could be seen as cells especially the smaller platelets. Never agitate box just before putting it on the instrument. Another source could be dried reagent spills or leaks. These can form salt deposits that create background electrical noise. Why would electrical noise influence results? We need to look at the counting chambers for this.

Copy from Coulter LH780 Manual
Here we have a typical counting chamber. The diluted sample enters the chamber and is draw through the aperture to the other side. If everyone remembers the coulter principle there is an external and internal electrode. When sample is pulled through the aperture there is a change in electrical current. This change in electrical current is sized and counted either as a RBC, Platelet or WBC depending on the aperture. So if there is salt deposits near the aperture this can create extra electrical impulses. Always clean all spills and leaks immediately. (I remember we had a blood gas instrument that was very sensitive to any static electricity. Depending on the clothing you were wearing you could introduce outside current to the reading. It was always a joke if you were having problems with getting the QC in that you had too much polyester clothing on.) Copy from Coulter LH780 Manual

All parameters have a “P” code Partial Aspiration Example 2 – Instruments have detectors to look at the closed-mode aspirated sample to see if it meets certain criteria. Depending on the instrument it will give you some type of code. In this particular instrument it shows as a “P” next to each parameter. This is know as partial aspiration error. Most instruments for the closed mode have a blood detector in the sampling valve. It checks to see if the blood coming in is the same concentration as that going out. If not you get this code. It is a way for the instrument to alert the technologist of possible sample problem when using the closed mode. This detector is bypassed when using the open or manual mode of sampling.

What can give this code? Sample could be clotted.
Hemoglobin is less than 4 g/dl Sample volume too low. The blood detector was turned off. Check your instrument manual. As for any problem your first step is to consult your troubleshooting guide. Here are some reasons you may have gotten this code/error message. Check the sample for clots. Even though this seems impossible I have seen patients with low hemoglobin but usually not the norm. To confirm if it is due to a low hemoglobin can run through open/manual mode. This bypasses the blood detector. If there were no clots and the result repeats manually then you technically can take the result. One last thing should do is make sure that the blood was not drawn through or above an IV line and was diluted out by saline. Check the volume in the tube. If less than 1 ml can’t be aspirated in auto/closed mode. Could be run in manual mode but should not. This sample would be diluted out and cause false low results. Blood detector was turned off. Since this is a way to detect questionable sample the instrument will not let you run in the closed mode with the detector turned off. There are certain procedure such as carryover testing where you need to turn off the detector. If you have turned off the detector it is very important to turn it back on. If the detector is on once again by consulting your troubleshooting it will give you steps to follow to determine if there is a instrument problem such as a bad detector.

Other Corrective Steps
Troubleshoot for leaks, kinks or plugs along the sample flow path. Ensure the aspiration lines are clean. Check for needle plugs. Call Service Hot Line Here are some other possible steps you may find in your troubleshooting section.

Get a 30psi Pressure High Error Here we have an error code for one of the pressure systems. This pressure on the instrument dispenses reagents from pumps, opens pinch valves and moves air cylinder shafts. It is controlled through solenoids.

Solutions Find the 30psi regulator and adjust to 30 +/-0.2 psi.
Rerun several bloods and compare to see if any difference. If unable to adjust or error occurs again call Technical Hot line. So checking with our troubleshooting manual we would look to see what we need to do. We are in luck this is one of the adjustable pressures so

Post-Analytical Control
Understanding your instrument results: Next we will look at post-analytical control. Post analytical looks at the results and determining if they are correct. Usually instruments have different flags, codes and messages. We will look at some of these.

Flags Can be a letter or symbol that appears to right of the result.
Manufacturer controlled Aspiration, Linearity, interference Laboratory controlled Critical range Decision rules Firs will look at flags need to recognize them on the instrument's printout. Usually with flags you will get a numerical result with the flag next to it. It will indicate a problem with the parameter. Examples are a symbols such as a “+” indicating the result is above the linearity. Can also be a letter such as the “P” flag indicating a partial aspiration as we saw in the previous section. The can also be added by the laboratory. The can be used to indicate either a low or high normal range and/or critical range. Some instruments allow you to input decision rules such as indicating a delta flag on the instrument printout.

Here we have examples. The one on the left shows the instrument flag
Here we have examples. The one on the left shows the instrument flag. These are from Coulter instrument. The other instruments have similar flags. Please check you manual for them. The middle one set by the lab showing the monocytes are above our upper normal range. The far right shows a combination of instrument and laboratory flags.

Codes Symbols that appear in place of results
Indicate beyond reportable range +++++ Vote-out of aperture Unable to calculate result Clogged flow cell Each instrument will have similar codes these are the ones for Coulter instruments.

Messages Usually manufacturer messages
Indicate possible abnormal cells, clumping, agglutination Used to decide on reporting of instrument vs manual differential or verification of measured results. Some will indicated possible abnormal cells, clumping, agglutination. When you did the validation of your instrument you should have included validation of the instrument flags, codes and messages. Policies should have been created when you needed to look at the slide and either perform a scan or manual differential. In your handout is a sample of a chart indicating what type of verification is required for the different flags, codes or messages.

I W Here are some Messages that you can see.

Patient Results Are the results consistent with the patient’s condition? Delta checks Can be set on instrument or LIS. Checks against previous result. Value set by laboratory. In addition to looking at any flags, codes or message on the printout you need to look at the patient results. You should ask yourself are these values what I would expect to see with this patient. For example on HIV patients they usually run low WBC. If suddenly you get a patient with an extreme high WBC you should wonder is this patient having some type of infection on top of their HIV or is there an instrument problem. For the patients that are getting blood drawn weekly you get to know what to expect. One way to monitor changes between visit is the use of a Delta check. Can anyone tell me what a delta check is? It is done either through the LIS system or the instrument where the patient results are compared to the last previous result to see if there are any significant changes to specific parameters. The laboratory decides on which parameters to monitor for change. Usually it is the WBC, HGB/HCT, MCV, MCHC and platelets. The % difference from the last result is also decided by the laboratory. As an example MCV is a very stable indices throughout your lifetime unless you have some major medical issue. You set a delta check to alert you if goes +/- 3 from previous test. A reasonable change in MCV would be if the patient got several units of blood.

H & H Check/Difference Hgb x 3 = HCT +/- 3%
If > +/-3 can indicate a problem. Instrument Sample Last type of message you can get is the H&H check. How many use this code? What is it? That is correct typically if you multiple the hemoglobin by 3 it should equal the HCT +/- 3. Can indicate instrument or patient problems. Since the parameters are measured in different areas of the instrument it can give you a start on where to look. If sample might have certain conditions such as agglutination either cold or warm. Also can alert you for a lipemic samples.

Exercise 2 Let’s do a couple exercises for Post analytical problems.

Post Analytical Example 1
Laboratory Results WBC - 8,900/uL RBC - 4,460,000/uL Hgb g/dl HCT- 40.7% Platelets - 56,000/ul MCV fl MCH pg MCHC g/dl RDW % Instrument Differential Neutrophil % % Lymphocyte % % Monocyte % % Eosinophil % - 1.4% Basophil % - 0.5% Flags WBC interference (*) Micro/Fragmented RBC Giant Platelets R (Review)-code on Platelets Platelet Clumps A 45 year old women had her blood drawn for routine testing. A CBC was run on a Coulter GENS instrument? What stands out on these results? Low platelets, all the flags What should be done? Make a slide and confirm results.

So here is what the tech sees when they look at the smear.
What is it? Answer: Platelet Satalitosis/Platelet clumps Couple reporting choices. First could try vortexing the purple top for about 1-2 minutes, make a slide then run manually on the instrument. This sometimes gets rid of the clumps. Second possibility is drawing a blue top. Sometimes the EDTA in the lavender can cause the clumping. You run it on your instrument and make a slide. If you see no clumping then the WBC and platelet count from the blue top can be used. But what has to happen to the results before reporting them? They need to be multiplied by 1.1 due to the dilution of the anticoagulant in the blue top. If none of these get rid of the clumping then should report out an estimate only no numerical result.

Here is what it looked like after vortexing
Here is what it looked like after vortexing. If you choose to use this procedure you will have to validate it. This can be done by running at least 20 samples that do not have any platelet clumping seen on the smears. After the initial run then you would vortex each sample for specific amount of time. We did 2 minutes. You would make a slide immediately and run it manually not closed mode through the instrument. You do it for all 20 samples than calculated the % difference before and after vortexing. You would want to see less than 10% difference in the results for the platelets. On patients with clumping what you will see on the post vortex is a decrease in the WBC and an increase in platelets. Once again you need to confirm with a smear. Why would you see a decrease in WBC? The clumps were counted as WBC. For reporting all results from the pre-vortex sample are reported except the WBC and Platelet and instrument differential. The vortex WBC and platelet replace the original if all clumping is gone. Of course if you need a differential on this sample it should be performed manually.

Laboratory Results WBC - 8,500/uL RBC - 4,870,000/uL Hgb g/dl HCT % Platelets - 356,000/ul MCV fl MCH pg MCHC g/dl RDW -12.5% Spun HCT – 44% Instrument Differential Neutrophil% % Lymphocyte % % Monocyte % - 8.2% Eosinophil% - 2.0% Basophil% - 0.2% Flags H-H difference check Alert (aH) on the MCHC The patient is a 54 year old man. A routine CBC was drawn in the out-patient department and it was run on a Coulter LH750. The technologist reviewed the instrument CBC, automated Diff and flags These are the problems that were noted – MCHC > 36.0 H&H Check Flag - H & H does not pass rule of three (three times the HGB should equal the HCT +/- 3) The laboratory policy is to spin a Hematocrit on any MCHC >36.0 The Spun hematocrit matches the hematocrit from the instrument. Therefore, the HGB is the incorrect parameter –

What is the Explanation ?
Spun Plasma The MCHC is > 36 The spun hematocrit matches the instrument It would appear that the Hgb is incorrect Lipemia will falsely increase the hemoglobin result Follow laboratory policy for lipemic samples It appears that the incorrect parameter is the Hemoglobin since the spun Hematocrit matches the instrument. Let’s look at the data that we have: Here is what the spun plasma looked like. The sample is lipemic. Lipemia falsely increases the hemoglobin result. What are your options? Do not report out the Hemoglobin and any indices with an H - MCH and MCHC due to the incorrect hemoglobin result. This is the easiest. Replace these parameters with a comment that the sample is lipemic and unable to report out these results. Perform a saline replacement procedure. We have included one in your handout. This is more time consuming. Run the hemoglobin on a HemoCue which is a POC instruments. (The hemoCue uses a different methodology for measuring hemoglobin) It will depend on your site’s policy which option you use.

Laboratory Result 1 WBC - 8,500/uL RBC - 4,870,000/uL Hgb g/dl HCT – 48.2% Platelets - 356,000/ul MCV fl MCH pg MCHC – 34.0 g/dl No flags Laboratory Result 2 WBC – 8,300/uL RBC – 4,000,000/uL Hgb – 16.2 g/dl HCT – 36.0% Platelets – 340,000/uL MCV – 90.0 fl MCH – 40.5 pg MCHC – 45.0 g/dl H-H difference check Delta Check on HCT and MCHC The patient is a 42 year old man. Two routine CBC was drawn a week apart in the out-patient department and it was run on a Coulter LH750. The technologist reviewed the instrument CBC, automated Diff and flags It looks very similar to the previous result that was lipemic These are the problems that were noted – MCHC > 36.0 H&H Check Flag - H & H does not pass rule of three (three times the HGB should equal the HCT +/- 3) We have Delta Checks on the Hct and MCHC The laboratory policy is to spin a Hematocrit on any MCHC >36.0

When we look at the spun hematocrit this is what it looks like
When we look at the spun hematocrit this is what it looks like. It shows that the sample is hemolyzed. A Hemolyzed sample would causes a decrease in the RBC count which in turn would affect the HCT result. Hemolysis would not affect the Hgb or MCV so it will cause an increase in the MCHC. It also affects the MCH result.

Laboratory Result 1 WBC – 6,300/uL RBC – 2,321,000/uL Hgb – 6.5 g/dl HCT – 19.5% Platelets - 250,000/ul MCV - 75 fl MCH – 28.0 pg MCHC – g/dl RDW – 9.8 Laboratory Result 2 WBC – 6,800/uL RBC –3,475,000 /uL Hgb – 9.5 g/dl HCT – 28.5% Platelets – 240,000/uL MCV – 82 fl MCH – 29.0pg MCHC – g/dl RDW – 13.4 Delta Check on Hgb, HCT, and MCV The patient is a 32 year old female. Two routine CBC was drawn a week apart in the out-patient department and it was run on a Coulter LH750. The technologist reviewed the instrument CBC, automated Diff and flags We have some differences We have Delta Checks on the Hgb, Hct, MCV, and MCHC Any possible explanations?

Here we have the RBC histogram
Here we have the RBC histogram. You can see there is a large population of RBC below 80.

Here we have a view of the slide.
So it confirms that we have a 2 cell population. How could we get such an increase in the H-H that quick? Most likely they got about 3 units of blood. Each unit of blood usually raises the Hgb by 1 unit and the Hct by 3.

Internal Quality Control External Quality Assessment
Analytical Control Internal Quality Control External Quality Assessment Now we will go onto the Analytical Control section of Quality Assurance It is broken into the Internal Quality Control External Quality Assessment Both are essential to ensure that the test are performed correctly and that their results are reliable.

Internal Quality Control
We will first talk about internal quality control. There is a lot to cover when you talk about Internal Quality control.

Internal Quality Control includes
Daily control specimen Commercial Control Patient Control XB Analysis – Moving Average Correlation/Comparison System Policy- QC and Troubleshooting With hematology you have several choices to use for internal quality control. They can be used alone or in combinations. Daily control specimen can have Commercial Another type of daily control you can have with hematology are patient control. Anyone use these? And my most favorite one to use XB analysis or moving average. If you are able to use this you can see within 100 samples that you may have an instrument problem. Along with these other controls you should have some type of Correlation/comparison system And of course with all of these you will need policy for both acceptability of controls and if they are out troubleshooting for problems.

Commercial Controls

Commercial Controls Assayed vs Non-assayed Introducing New QC Lot
Establishing QC Ranges Most regulatory agencies have standards how many and often controls should be run. Usually it is at least two levels run in a 24 hour period or on days instrument is used. Typically for hematology there are three levels of controls. Again the number of time they are run will depend on a number of factors one being the number of specimens processed. If you only run around 50 per day than once a day may be enough but if you run 1000 samples than multiple times would be better. You don’t want to find out after running 500 samples that a problem has occurred. That would be a lot of patients results you would have to re-confirm. For commercial controls there are two types you can get the assayed – with already established means and ranges and the Non-assayed where you will establish you own. I strongly suggest to go with the assayed. Ideally when you get a new lot you will still have your old lot to run in parallel. I know with many of our international labs this doesn’t always happen. You are just happy that you get them a day or two before your other ones expire or run out. We will go through the steps of lot-to –lot comparison. After the initial parallel running and the controls are now in use each laboratory should establish their own means and ranges. The values stated on the assay sheet provided by the manufacturer should be used only as guides in setting the initial control limits for testing the new lot. The observed mean should fall within the range published but does not have to match it perfectly. I will also go over a simplified version on how to establish your own.

Lot–To-Lot Correlations
Set up new QC files for each control level of the new lot. Verify the new lot by running each level of control three times in its respective file. Ensure that the mean values of the control runs fall within the ranges published on the manufacturer assay. Run each level twice a day for 3-5 days to calculate new mean values for each analyte. Compare the calculated mean values for each level to the range specified on the manufacturer assay sheet. If the calculated mean is within range, enter it as the expected mean. So for the lucky ones that do get their new lot before the old is gone you should be doing a lot-to-lot comparison or parallel testing Here are the steps: #1 This is where you can use the mean and range that came with the set of controls. #3 They don’t have to match exactly just be in the range. So why do we do this first? You want to ensure that the new lot is good. If they fall outside the range first you would try a different vial but if that one is out too you will need to contact with your supplier and get a new set sent to you. #4 once you have seen that the controls are usable you will #5 compare this calculated mean to manufacturer’s #6 Once the values are updated in the QC files, these values will be what you use when you start using the new lot until you establish your own mean/range.

Establishing Means and Standard Deviations
Analyze the control(s) a minimum of 20 times across several days. Take the average of these runs. The average should be within the range state on the assay sheet. Calculate a two Standard Deviation range from your results. Incorporate this SD range around your new mean and monitor. The mean and SD values should be periodically recalculated during the life of the new lot. So once your are ready to switch from the old QC to the new lot you will first use the mean established in your lot-to-lot. This will be your starting point to establish your own mean and standard deviation Here are the steps. #3 This mean will be considered as the “new Mean” Most hematology instruments have quality control files that will calculate these means and ranges automatically. What I have just gone over is a very simplified version. SMILE actually has another PowerPoint we have done in previous years called “Improving the Sensitivity of QC Monitoring: Taking the leap from manufacturer's to established QC ranges. It goes into detail on how to utilize historical CV, EQA CV and Manufacturer’s CV in order to develop useful quality control ranges for your particular instrument.

Factors to Consider for Means
Some hematology parameters, such as MCV, Platelets and RBC will start to change over time. MCV will increase. RBC values can decrease. Platelets values will increase. Mean, SD, and CV should be evaluated monthly. Due to the make up of hematology controls over time there will be normal breakdown of cells that will show up particularly in the MCV, RBC and Platelets. The RBC will start to take on fluid so you will see the MCV increasing over time by as much as 2 units. Another things that can occur is the actual rupture of the RBC due to the swelling. In this case you will see a decrease in your RBC. Because you now will have RBC fragments they can be counted as Platelets so the values will increase. Your QC should be evaluated monthly to ensure continued acceptable performance. When adjusting QC it is important to include all valid data collected since the material was put into use. Only omit data points caused by know operator or instrument error. Don’t delete points if just outside your range (2SD). If you eliminate them you are excluding true variability of controls. If the mean changes significantly, such as more than 1 SD than you should check the calibration and adjust as needed.

Exercise 3 We will not do a couple of exercises establishing means and ranges for your controls. The first one will take into consideration the increase of MCV due to swelling.

QC Example 1 Know variation of mean 2 fl Initial MCV mean – 84 fl
Historical 2 SD – +/- 4 fl Use half known variation accommodate change 1 fl What should the mean be? 85 Established range – fl Cumulative mean at end of product life should be 85. Through using historical data on previous controls our lab has established that the normal swelling of the RBC can increase the MCV result by 2 fl. First we will look at establishing MCV means. Our initial MCV mean is 84. Our laboratory has established through previous QC that our historical 2 SD is +/- 4 fl. As I have said we have noted a 2 fl increase through the running period of our control. So to compensate for this increase it is acceptable to raise the mean by half this change to accommodate this known rise. So if your initial mean was 84 what can we set our compensated mean to? 85 Using our 2 SD what would our range come out to be: With these settings our values will start below the mean then rise through it and finishing above the mean. At the end of the product life the cumulative mean (mean calculated with all the data points run) should be around 85. If start seeing this average above then could indicate instrument problem such as calibration needed. Again by using historical data for RBC and Platelets changes you can establish the RBC means slightly below the initial mean and for the platelets slightly above the initial mean. As the cells fragment the RBC will start to fall while the Platelets value rises. Again by the end of the product life your cumulative mean should be close to they original mean.

81 80 82 84 85 86 83 87 88 89 90 85.20 Here we have an example of what the LJ chart might look like with our previous exercise.

QC Example 2 Yes mean within the expected range.
Manufacturer RBC mean – 2.29 x 106 Manufacturer RBC Range – x 106 Calculated RBC mean – 2.35 x 106 Is this mean valid? Our 2SD = 0.15 x 106 Calculated RBC range – x 106 Yes mean within the expected range. So here is the manufacturer’s RBC mean and the range from the package insert. We perform out testing to establish our own mean. Is it a valid mean? Yes within the range. Now we will figure out our range. We calculated our 2 SD and it is 0.15 So what is the range? So can we use this range? Yes as long as our mean is within manufacturer’s range does not matter if our range is exact.

QC Example 3 Is this mean valid? No below the expected range.
Manufacturer Platelet mean – 528 x 103 Manufacturer Platelet Range – x 103 Calculated Platelet mean – 402x 103 Is this mean valid? No below the expected range. Here we have the manufacturer’s mean and range for platelets. Our calculated mean is Is this mean valid? No, our calculated mean is below the manufacturer range so this would make our mean invalid. If this was your lot-to-lot check then maybe it is just a bad vial. Try repeating with another vial. If continue see problem hopefully you have time to try to get a replacement set. If your original lot-to-lot was in and this is your result after establishing your own mean need to investigate why not within range. Look at the data to see if there are any points that are questionable such as the wrong level run in the wrong file. If can eliminate any points then may need to get new lot in. If you are using 3 different levels of controls technically depending on your regulations could continue to use other two if within valid range.

Commercial Control Monitoring
Levey-Jennings Charts So we have done the calculations to create our means and ranges how will we monitor them?

Levey-Jennings QC Charts
The Levey-Jennings QC Chart is the most commonly used chart used to show the results of a control for a specified period in a graphic form. The chart depicts the position of data points relative to the assigned mean. It also includes our SD. Here we see 1 and 2SD range. Also you can see the outliners. What are Outliners - Outliers are discrepant values which do not agree with the pattern of the rest of the points. They may be due to mistakes or they may be a significant finding. Above we see a big jump above and below the mean. Statistically 1 out of 20 points outside the limit is normal. Usually rerunning the sample the result will be in this time so you can assume that it was just a normal outliner. It is when you start seeing patterns that you have to worried about it. We will look at three patterns you may get with your LJ charts

Trend First we will look at a Trend. A trend occurs when five or more values show a gradual increase or decrease. The values do not have to be out of your acceptable range but this pattern can indicate that a problem exists. Because hematology controls are cell-based, some trending in sizing parameters can be expected. What parameters did I mention can increase or decrease over time? The MCV, RBC and platelets. What you are looking for is excessive trending.

Shift Next we have a Shift. This is when there is a sudden change in control results from one run or day to the next. A shift does not always mean that a problem exists. What are some reason you might get a shift? Instrument calibrated. Service performed.

Bias Finally we have a bias. A bias is when your control starts running on one side of the mean. It can occur with just one or all of your controls. If it is only one of your control can indicate a problem with just that level. If it is across all the controls than can indicate an instrument problem or the need to calibrate.

Here we have a typical LJ chart for a hematology instrument.
We have the original means and upper and lower ranges. Here we have the cumulative range. Let’s look at a couple of the analytes. If we look at the WBC we see that the cumulative shows a slight negative bias. Here with the Hgb we see that the whole time there was a positive bias. And with the platelet it starts slightly below the mean, on the mean and toward the end above

QC Policy Each laboratory should adopt QC rules.
Establish policy/corrective action for controls that are “Out”. Documentation is important. Establish policy for Trends, bias and shifts. Establish when calibration are required. With all Quality Control you need to create policies for acceptable and unacceptable results. Need to adopt QC rules. Of course the ones that most of us know are the Westgard rules. I will show you two typical ones in our next slides. Again on our Resource section on pSMILE.org we have several documents going over Westgard rules. What do you do if the control is “Out” – Beyond your established range. If control is “out” first rule would be - Do not run any patient samples until determining what caused the control to be out, and solve the irregularity. Everything should be documented such as the date of occurrence, what is out such as WBC low control, the lot# and expiration date. What action performed such as rerunning and repeat in range. Should include initials of person performing the corrective action. All of these occurrences need to be reviewed by the supervisor. Same thing for Trends, bias, and shifts. Need to establish when and what to do if you notice trend, shifts or a bias. Usually with hematology instrument unless there is major service done the calibration is very stable and usually done every 6 months. When you are deciding if a calibration is needed you look at all levels of QC. If each level is showing the same consist problem such as all running a bias below the mean could indicate the need to re-calibrate. Depending on the instrument you may be able to perform the calibration yourself or it may require a service call.

Here is a diagram showing they typical Westgard rule chart that can used for 3 control levels. It uses a combination of decision criteria, usually these 5 different control rules to judge the acceptability of an analytical run. They are 1 3 S – run is rejected when a single control exceeds the mean by +/- 3 SD 2 of 3 2S – run rejected when 2 of 3 controls exceed the mean +/- 2SD R 4S – when 1 control in a group exceeds the mean +/- 2SD and another control exceeds the mean in the other direction by 2 SD run is rejected. 3 1S – run rejected if 3 consecutive control values on one side of the mean and further than 1 SD from the mean. It can be one control across 3 consecutive runs or all 3 controls on one day. 12X – run rejected when there are 12 consecutive values on one side of the mean. Can be either one control for 12 runs or between the 3 controls for 4 runs.

Here is another Westgard combination
Here is another Westgard combination. It is usually used with 2 controls such as in chemistry. Here it starts out with the 1 2S rule – where one control is out +/- 2SD. This should be used as a warning rule to look to see if there are any other rules occurring. As you can see if you get this rule then you need to get at least one of the other rules to reject the run. As I said previous statistics show that there can be at least one outliner for every 20 runs. Westgard Multi-rules are used to reduce costs while maintaining a high level of certainty that your analytical process is functioning properly. It helps to decrease the number of false rejection without compromising quality.

Policy Example If control out +/-2SD and a second Westgard rule is also seen Rerun the control. If another vial of control is available use it. No more than 2 control reruns from same vial or new vial should be made. If control still out begin troubleshooting do not report patient results. Check maintenance log Check reagent Check calibration date Here is an example of what a QC policy could look like.

Troubleshooting Guideline
Should have a policy on how to perform trouble shooting for out of range results. Instrument vs sample problem Resolve problem before running patients Can create a checklist . Can also include a Corrective Action Flowchart . In addition to your QC policy every laboratory should create policy on how to perform troubleshooting when their QC is out of range. Part of your investigation will be establishing whether it is a sample problem vs an instrument problem. If you have two similar instrument this is easy. You just can run the questionable control vial on the other instrument. If get similar results than sample problem but if results on second instruments are within range than it points to an instrument problem. If only one instrument than can try opening a new vial and running to see if the results differ or are the same. Should troubleshoot the problem and remedy the situation prior to running patient samples. It is good to have a checklist for your techs to follow. I have include an example of a checklist in your hand out. Another helpful item could be a flowchart which is also included in your handouts.

Exercises 4 Now I will show you some LJ charts and I want you to tell me first if you think there is a problem vs random outliner. Also it there is any specific pattern.

LJ Example 1 Here we have half a month of points on our abnormal high control for hemoglobin. We have an assigned mean of 17.1 and an expected range of +/ Do you see any problem? Negative trend. If seen in only one control what could be the possible problem? Vial. If see in new vial? What would you do? Document and investigate. Here is where knowing how the parameters are measured on the instrument come into play. How are Hgb measured? Diluent flows into the Hgb cuvette to take a “blank reading”. Depending on the instrument then either patient’s WBC sample or a separate Hgb sample drains into the hemoglobin cuvette. For both readings a beam of white light from a lamp goes through this cuvette then through an optical filter that has a wavelength of 525 nm onto a photocell. This reading is sent to the analyzer. The blank reading is subtracted from the blood reading to get your final result. So what could affect this lamp reading? If the lamp is fading this would decrease the light which could result in low hemoglobin readings. Another reason for lower values is if bubbles are getting into the cuvette with the blank reading. This would make its reading higher thus decreasing the sample reading.

LJ Example 2 Here we have a LJ chart for platelets normal control. The mean was set at 230 with an expected 2sd range of 25. What do we see happening after the 7th day? A shift What would be some things to look at? Check for correct lot number? When was the vial opened? Is vial expired? Was it properly stored? When was instrument last calibrated? When was last service? This is platelet so as mentioned before there is a tendency to see some shifting to the positive side due to RBC breakdown. This pattern is more of a abrupt shift not a trend. Again you need to have a policy at what point does this type of change warrant corrective action.

LJ Example 3 Here we have all three levels for the RBC.
What do you see. See a bias on all three. We see that the points are being reproduced showing the instrument precision is good however the results are not accurate. They are all on the low side of the assigned values, a negative bias. First should make sure no instrument problem exist such as dirty aperture. If you have a build up of protein on the aperture can lead to lower counts. Check to see if maintenance has been done. For coulters would need to periodically bleach the apertures. If not an instrument problem then could indicate that a calibration is required.

LJ Exercise 4 Normal variation around the mean

LJ Example 5 Beginning looks good then for several points runs a bias, then have one dramatic shift up then back down. Bias could be the vial. What could be explanation for the dramatic shift? Chance outliner. Wrong control.

Retained Patient Controls Now we will look at a second type of internal QC

Retained Patient Control
Two-three patient samples with specific parameters. Can be used over a 24 hour period. Cost efficient. Can be used to detect systematic error. Transferable between instruments and modes. So another type of QC you can run is the retained patient controls. Do you use retained patient controls as part of their QC system? What is it? Usually consist of 2 -3 patient specimens pick from morning run. Preferably newly drawn blood, not sitting around for a long time. Should be a full draw because will be running it several times maybe on different instruments and/or modes throughout the day. Can be used over a 24 hour period. There is no significant changes in major parameters for 24 is properly stored Why use them? Cost efficient. Can be run at closer intervals than commercial controls. Will allow you to have smaller batches of samples to repeat if find there are instrument problems. They can detect systematic errors Good way to correlate between different instruments and modes. Once again cost efficient.

Just like commercial controls should have
Sample Type WBC HGB Control 1 x 103/mm3 mg/dL Control 2 x103 /mm3 mg/dL Parameter Control 1 Control 2 WBC RBC HGB HCT MCV PLT ± 0.6 ± 0.14 ± 0.3 ± 2.0 ± 40.0 ± 1.4 In my laboratory we did 2 retained bloods using the WBC and HGB parameters as our guide. One had results in the normal range while the other had a higher WBC and lower Hgb. Don’t want anything extremely abnormal such as low or high platelet count. Abnormal indices. Usually monitor the parameters that are measured by the instrument such as the WBC, RBC, HGB/HCT, MCV and Platelet. You want the measured parameters to help detect the systematic errors of your instrument. For the ranges can use historical SD to set up them up. If don’t have them any historical ranges then can use the evaluation criteria used on either CAP or OWA in the beginning. Over time you should collect your own data and calculate your own ranges. As you can see for the WBC there is a difference in range between the two levels. Just like commercial controls should create a policy for “Out liner” and corrective action Just like commercial controls should have policy for “Outliners and corrective action.

Control Decisions If Patient Control outside of acceptable range rerun
Still out Results still the same on back-up instrument Results acceptable on backup instrument THEN Proceed with patients Use back-up method Sample deterioration proceed with patients Indicate instrument problem, use back-up instrument. Inform QC tech or supervisor Here is an example of a policy for our retained patients controls when we would have a parameter out of range.

Exercises 5 So now we will do a couple of exercise looking at patient control

Patient QC Example 1 Original Result Scheduled rerun
WBC – 5.5 x 103 /ul RBC – x 106 /ul Hgb – 12.4 g/dl Hct – 36.5 % MCV – 85 fl Plat – x 103 /ul WBC- 5.9 x 103 /ul RBC – x 106 /ul 12.0 33.5 % 84 fl 268.0 x 103 /ul Here we have the first run of the high patient control 4 hours later we run it again and get these results Let’s look at the difference on each WBC /- 0.6 within range RBC / out of range Hgb /- 0.3 out of range Hct /- 2.0 out of range MCV – / within range Plt – / within range So what should we do? Repeat it

Patient QC Example 1 Original Result Repeat Result WBC – 5.5 x 103 /ul
RBC – x 106 /ul Hgb – 12.4 g/dl Hct – 36.5 % MCV – 85 fl Plat – x 103 /ul WBC- 5.8 x 103 /ul RBC – x 106 /ul 12.0 33.0 % 85 fl 260.0 x 103 /ul We repeated it and this was our results Let’s look at the difference on each WBC /- 0.6 within range RBC / out of range Hgb /- 0.3 out of range Hct /- 2.0 out of range MCV – /- 2.0 within range Plt – / within range So looks like the RBC parameters are still out after the repeat. So according to our last slide we would then run it on the back-up instrument

Patient QC Example 1 Original Result Back-up Result
WBC – 5.5 x 103 /ul RBC – x 106 /ul Hgb – 12.4 g/dl Hct – 36.5 % MCV – 85 fl Plat – x 103 /ul WBC- 5.8 x 103 /ul RBC – x 106 /ul 12.1 33.5 % 85 fl 265.0 x 103 /ul On the back-up we get this Let’s look at the difference on each WBC /- 0.6 within range RBC / out of range Hgb /- 0.3 within range Hct /- 2.0 out of range MCV – /- 2.0 within range Plt – / within range So both the RBC and Hct have similar results So this would show what? Sample deterioration. We would be allowed to continue to run patients. As long as one of the retained patient controls is usable just run the one the rest of the day. But if both aren’t useable due to deterioration or running out of sample than 2 new samples should be picked for the rest of the day.

XB- Moving Average We will look at the last type of Internal QC you can do in hematology

XB – Moving Averages Cost effective quality control method.
Allows for continuous monitoring of system performance. Uses patient samples in conjunction with other controls. Created by Brian S Bull. Algorithm evaluates RBC indices. Must run either 100/day or 400/week Last Internal QC will talk about is XB – Moving Averages How many use these on their instrument? XB analysis is another cost effective quality control method allows for continuous monitoring of system performance using patient samples in conjunction with commercial and patient controls. Created by Brian S Bull. It uses a complex algorithm that evaluates RBC indices which are typically stable for a individual patient, from day to day, and stable for patient population over time. They are considered as an added support to a lab’s QC program being very effective if the lab has a volume of > 100 samples/day or 400 per week. It uses small batches of 20 samples to calculate a point for each parameter.

How to Set Target Values
Start with Bull’s values: MCV = 89.5, MCH=30.5, MCHC=34.0 Run 400 bloods or one week’s worth Calculate the mean (target value), SD and %CV %CV has to be </= 1.5% Target value should be within 3% of Bull’s The values must be physiologically possible Upper and lower range set at 5% of target First set to Bull’s values Next run either 400 bloods or a week’s worth. Again you need to run at least 100 per day or 400 /week Want to calculate your own values. Need to make sure the %CV is </= 1.5%. When monitoring your QC CV% monitors your instrument precision. It has a little different use with XB, if your CV is higher than 1.5 it means you don’t have a random enough population. You need to look back through your runs. Did you have a big batch of specific patients such as babies, oncology patients, renal patients. Depending on if this will be your usual population of samples it may mean you can’t use XB to monitor your instrument. If it was an unusual batch of specimens the you can these results and try calculating your values again but remember you still need to have at least 100/day or 400/week. Your target values should be within 3% of Bull’s target values And most important of course it has to be values have to be physiologically possible. In beginning can set the upper and lower range at 5%. After using it for several months re-evaluate to see if you can decrease it down to 3%.

Reason Why Moving Average to be Out
Non-random population Instrument problem Calibration Example would be got a large batch of bloods from specific clinic such as oncology, renal or even children Actually problem with the instrument. Later we will go over some examples. I have included a SOP and flow diagram for you to follow. Last you had to calibrate your instrument and this could have changed the target values. You would have to run evaluated the target value like you did in the beginning.

Exercises 6 Now we will do exercises covering the different types of Internal QC.

Parameters used in XB MCV – usually direct measurement
MCH = Hgb x 10/RBC MCHC = Hgb x 100 / HCT (Usually Calculate) HCT = RBC x MCV/10 Next will look at XB moving troubleshooting. First lets look at the three parameters used. MCV usually is a direct measurement especially in Coulter instruments. So things affecting the RBC aperature would affect this parameter such as protein build-up, cracked or leaking aperature. For the MCH you would look at both the RBC aperature and the Hgb section of the instrument. For MCHC would look at the three parameters – Hgb, RBC and MCV For Sysmex instrument the calculated parameter technically is the MCV and the HCT is a measurement. For our examples we will be using the direct measure MCV and the HCT as a calculation.

How to troubleshoot with XB
MCV Involves RBC aperture Tell if problem with sizing or flow problem MCH Involves Hemoglobin sample which is part of WBC sample Involves RBC count MCHC Involves all three parameters. So how can these three indices tell you what is going on with your instrument? First the MCV is measured in the RBC aperture. If the MCV starts being larger then it telling you the aperture is getting smaller. This in term will cause less amount of sample flowing through the aperture causing lower RBC .The most likely cause is protein buildup. On the other hand if the MCV is getting smaller than the aperture size is increasing. This can be caused by cracks in the aperture itself. This will increase the flow of RBC so the count will increase. For the MCH It involves the HGB portion which is also the same sample for the WBC count. So this will give you insight to that section of the instrument. It also involves the RBC count so once again you will be able to detect problems that can either increase or decrease the count. Usually problems with the RBC only is due to the vacuum used to pull the sample through the aperture. For the MCHC It involves both the RBC and WBC/Hgb systems. Use it in conjunction with other two indices to see the common problem.

XB Troubleshooting Policy
Moving Average Acceptability If Then Moving average parameters agree within the established limits set Proceed with test of patient samples. If 5-6 batches fall outside the limit set and cannot be explained Hold testing of patient samples and run on backup until investigation complete Investigation Here are examples of XB troubleshooting policy

Example 1 The following exercise is using the flow chart for a Coulter instrument where the Hct is the calculated parameter while the MCV is the direct measure. A similar flow diagram can be created for other instruments substituting where the MCV is the calculated while the Hct is the direct measure.

MCV MCH MCHC See both MCV and MCH on the rise and the MCHC is steady

Yes Yes Yes Yes No Yes Yes No No Bleach aperture
Do MCV and MCH return toward mean within 5 points? MCV Hi MCH Hi MCHC OK Yes Yes Yes Due to patient population Continue to run Yes No Protein build-up in aperture Yes Results OK? Yes Bleach aperture No No So let’s look at one pattern you can find with your three indices. First we want to see if we continue for at least 5 points this pattern. If comes back to mean then it was a non-random population. If continue this pattern then cold be an instrument problem. With this pattern we know the MCV is affect and it is measure on the RBC side so most likely it involves that part of the instrument. Something that could increase the MCV is protein build up on the RBC aperture. So first look at the maintenance to see when last time was bleached. If it has been at least a month since last perform than do the bleaching. Once you bleach you should then pick 10 samples that were run before the bleaching to see if there are any noticeable changes to the RBC and MCV. If there is than should run at least 60 – 100 samples to see if the MCV and MCH start back toward the mean. The all should return toward mean within 5 points if not then call for tech support. If the instrument had just been bleached or the post-bleaching results did not solve the problem then you would need to call for Tech support. Now we will look and see why we would get this pattern. Call for Tech Help

Indices Pattern: MCV MCH MCHC OK
Make the aperture diameter smaller Decrease number RBC getting through Increase MCV MCH = Hgb x 10/RBC Increase MCH MCHC = Hgb x 1000 RBC x MCV No change If you had protein buildup what would happen to that aperture – smaller diameter – cause the RBC to appear larger so get increase in the MCV. If aperture is smaller then will decrease the number of cells getting through the aperture so RBC be decreased. So looking at the other two indices how would that affect them? MCH would be increased MCHC would stay stable Why? Again look at the calculation. The RBC would be smaller but MCV larger so cancel each other out.

Example 2

MCV MCH MCHC Here we have the MCV steady but the MCH and MCH are low

Hgb RBC Yes Yes Yes No No Yes Adjust to 6.00 +/- 0.01
MCV OK MCH Low MCHC Low Yes Either Low Hgb or High RBC Problem Check Controls for Bias or Trends Check for bubbles in Hgb cuvette RBC Yes Yes Check low Vacuum 6.0+/-0.01 Call for Tech Help No No Adjust to /- 0.01 Results OK? For this pattern the MCH and MCHC have problems. The common parameters are either the Hgb or RBC. First we should look at the QC charts to see if any trends, shifts or bias has occurred with either. So if see any negative biases, shifts or trends in the Hgb then check Hgb cuvette. If there appear to be bubbles in it then need tech help. If see positive bias, shift or trend in the RBC QC then could be a vacuum problem. We need to check the pressure involve with moving the RBC through aperture. On our instrument it is the 6.00 vacuum gauge. First look to see if within the limit. If it is then we will need to call tech support. If outside the limit then we can adjust. We would run 10 previous patient samples to see if there are any differences in the RBC. If we see they have decreased then we can continue to run to see if XB returns to the mean. If it doesn’t return within 5 points then we need to call for tech support. Yes Continue to Run

Indices Pattern: MCV OK MCH MCHC
Bubbles in cuvette Increase Blank Reading Decrease Hgb MCH = Hgb x 10/RBC Decrease MCH MCHC = Hgb x 1000 RBC x MCV Decrease MCHC MCV No change Possible H&H Check First we will look for the hemoglobin problem. Most instruments take an initial reading in the hemoglobin cuvette without any blood called the blank. If you have bubbles in there it will cause the reading to be high. Now when the specimen is read it reading will be decreased. The two indices that this would affect is the MCH and MCHC. Easy way to remember is anything that has a M in it. So for MCH and MCHC will cause it to decrease. For MCV would not affect it stay the same Should start getting H&H checks because hgb x 3 not = HCT

Indices Pattern: MCV OK MCH MCHC
Low vacuum increased Increase RBC MCH = Hgb x 10/RBC Decrease MCH MCHC = Hgb x 1000 RBC x MCV Decrease MCHC MCV No change Possible H&H Check If it was the vacuum then it would affect the RBC. A high vacuum would pull more RBC through the aperture. So for MCH and MCHC will cause it to decrease. For MCV would not affect it stay the same Should start getting H&H checks because hgb x 3 not = HCT

Example 3

MCV MCH MCHC With this one the MCV is rising, the MCH is stable and the MCHC is dropping

Continue to Run Yes MCV HI MCH OK MCHC Low Yes Has diluent been changed within 12 hours Yes MCV and MCHC return to Normal Possible Frozen Diluent, Change High MCV Problem Before we start this one I have to say this pattern would only be seen if the laboratory was in an area that temperatures below freezing. So let’s at this pattern. Like always should look at the QC chart to see if you notice any increase in the MCV and decrease in the MCHC. We check first if we change the diluent within the last hours depending how many bloods are run in a day. If it had and it is cold season could be at one point in time the diluent got frozen. We should change to another cube. We can continue to run and if return within 5 points then it was the problem. If not then it is a high MCV problem call for support. If the diluent has not been changed or it is the warm season then it could be a high MCV problem. Need to call for support. Now we will look and see why we would get this pattern. No No Call for Tech Help

Indices Pattern: MCV MCH OK MCHC
Frozen Diluent Thawed will separate into two layers Hypotonic on top Hypertonic on bottom When get to top layer cause RBC swell MCV Increased MCH = Hgb x 10/RBC No change MCHC = Hgb x 1000 RBC x MCV Decrease MCHC I sure that this usually isn’t a problem but if the reagent does get frozen in transit can happen. In US where can get sub zero temperature can see it happen. The truck comes and leaves the reagent on the loading dock it doesn’t get picked up and sent to lab for several hours. What will happen it will freeze and when it thaws out it will thaw in layers - top will but hypotonic and the bottom layer will be hypertonic. At first the tubing will be drawing up the hypertonic solution so no change to any of the parameters but as it gets to the hypotonic will cause the RBC to swell increasing MCV What about the MCH not affected What will happen to the MCHC – on instruments that use that calculates the HCT then MCV in the denominator will decrease it Solution – always roll the boxes on arrival. Don’t want to do it just before opening – Why – introduce bubbles.

Correlation/Comparison
Blood count to blood film Blood count changes to clinical events Instrument to instrument Also include in internal QC should be correlation and comparison What type of correlation should there be: Need to correlation the instrument results to those on a blood film. If you get low platelet result you need to confirm it on the slide. Want make sure that it is truly a low count and not a false positive. As we saw previously under the Post analytical section on the example with a low platelet it could be to clumping. When validation was performed it should have included instrument differential to manual differentials correlation. It is important to correlate instrument flags, codes and messages to the blood smear. Your site should create a chart for when the results need to be check by a blood smear examination. It should include when you need to perform just a check on numerical results and when to perform manual differentials. In your handouts is include an example of such a chart. As I had said previously this is another area that should be validated when bringing a new instrument into use. Also need to have correlation between your patient results over time. This is where if you use delta checks come into play. If have a change say in the H&H does it correlate with the patients condition. Has he had blood lost, is he getting drugs that could affect RBC production? (Whether you think about it all technologist are detective. We are given a set of clues and with it we can actually diagnose. We see the results before any clinicians. If I get a lavender from a women in the OB ward and a half hour later get a new one and the platelets have decreased by half I will check that smear to confirm the result but also to see if I see Schistocytes. I also will be looking to see if any coag had been run. So if I see the consumption of platelets, increase in PT, PTT and decrease in Fibrinogen with mod-numerous Schistocytes what am I going to think? – This women is going into DIC) Also if you have multiple instruments need to perform correlation between them to insure getting similar results. Should also include correlation between modes – closed to open.

Factors to Consider with IQC
Type of instrument – if fully automated. The size of the lab. The level of training of your staff. The number of specimens handled each day. Dayshift vs 24 hour laboratory. Country’s own regulations. Accreditation requirements. Of course the amount of internal quality control depends on several factors. It will depend the, type of instrument, the size of the lab, the level of training of your staff, the number of specimens handled each day your country’s own regulations Any accreditation requirements

IQC Recommendations A three level commercial control along with a retained patient control. If patient numbers permits add XB to IQC Commercial controls Two levels at beginning, middle and end of shift Retained Patient Controls At equal times between commercial controls If your laboratory only has a dayshift then recommend 3 level QC with retained patient If patient numbers permit add the XB Suggestion for running the commercial controls. Suggestion for running patient controls.

QC Schedule Example 8:00 AM Commercial Controls level 1 and 2
10:30 AM Retained Patient Controls 1:00 PM Commercial controls Level 2 and 3 3:30 PM Retained Patient Controls 6:00 PM Commercial Controls Level 1 and 3 Here is a schedule for a lab running only a 10 hour dayshift. If your laboratory has more than a day shift then adjust the running of the commercial controls. Additional patient controls runs can be added to space them out. The major factor in deciding how often to run any control is the number of patients run. You want the least amount of patient results to be compromised. The more samples between controls the more you may have to repeat if you detect instrument problems.

This picture goes with the freezing diluent just a typical winter in Baltimore

External Quality Control
Now I will talk about External Quality Control the other part of the analytical process

Why need EQA To detect errors not detectable by internal controls.
Ensures harmonization between laboratories. Used recognize systematic errors. Lab Result – Mean/SD = SDI You ask how can this be. I have been running my QC, it has been in the entire time, they are within the manufacturer’s ranges, no problems. Well just because they are in those range doesn’t ensure your instrument may be running higher or lower that other laboratories with the same instrument and QC lot. This might not be as important to some sites where you are the only laboratory around but for patients that may travel between laboratories it is important to know if the change in their results is clinical or due to instrument differences The results can help recognize systematic errors through the use of the SDI – Standard Deviation Index. When we do your reviews we use this SDI to evaluate for Biases, Trends and Shifts

Bias –two or more specimen’s SDI are > 2.0
Shifts –All specimen’s SDI in single event > 1.0 on opposite side of previous event Trends – SDIs increase progressively in one direction for three EQA events away from mean When we look at EQA results the terms bias, shifts or trends have slightly different meaning than when looking at QC charts a bias is indicated when two or more specimen’s SDI in a single EQA event are > ± 2.0 as seen with this ex. This could indicate that your instrument may need adjustment of the calibration or laser. A shift is indicated when all the specimens in a EQA event are at least 1 SDI on the opposite side of the mean from the previous event as seen here with this ex May be seen if you had service – parts changed or adjustments made. trend is indicated when the SDIs increase progressively in one direction for three consecutive EQA events away from the mean. If the decrease toward the mean this is a good thing Could indicate a part such as a lamp wearing out, may need replaced.

SDI Interpretation <1.0 = satisfactory performance
= still acceptable but borderline = requires review of techniques and check on calibration >3.0 = defect requiring urgent investigation A chart that you can use to interpret deviation index is

Exercises 7

EQA Example 1 MCV Here we have results from a hematology survey.
Looking at the chart can see we had MCV SDI going from negative to positive bias with the last round having all 5 results >2.0 bias with three of them out. This shows a upward trend. The investigation noted a positive bias when EQA was run. The sites uses manufacturer’s ranges . RBC QC had negative bias and MCV had positive bias The site performed an investigation and this was their cause of their investigation And here we see on their next round that we have much better results.

EQA Example 2 Instrument Differential
This example is on a Coulter instrument where we are having problems on the instrument differential. As you can see we have unacceptable results and SDI > 2.0 SDI for the neutrophil for multiple samples, on the lymphs

And also more on the basophil and eosinophil results.

INVESTIGATIVE ACTIONS AND ROOT CAUSE: Briefly discuss what actions were taken in this investigation and what you believe is the possible cause. 1. We checked to find out whether there were clerical errors and found out that there was none. 2. After replacing the flow cell, the EQA specimen which failed were re-run and all were within range. The cause of the EQA failure was a faulty flow cell in the CBC/Differential Analyzer. This was confirmed because after replacing the flow cell the EQA samples were re-run, all of the values that had previously failed now fell within acceptable limits of intended results. The site used Manufacturer’s ranges. Their investigation said no problem with their QC. The site did an investigation and found that the instrument had a faulty flow cell. When it was replaced the results were in. Their Internal QC showed slight biases but not enough to realize there was a instrument problem.

EQA Example 3 Here we have results from a Sysmex instrument.
This survey was delayed in shipping so it caused it to have integrity problems. It would be easy to just look at all the unacceptable results and say nothing wrong with instrument. It was just integrity problem with the survey, but you need to take a closer look at the parameters. In particular the Hemoglobin For the hemoglobin reading the RBC are hemolyzed in order to be read by the instrument. So it would not matter if the sample already has hemolyzed RBC from the long delay still should be the same reading. You can see that there is a positive bias running on the hemoglobin. So instead of disregarding this survey the site should investigation the bias in their hemoglobin.

CV is It was just integrity problem with the survey, but you need to take a closer look at the parameters. In particular the Hemoglobin For the hemoglobin reading the RBC are hemolyzed in order to be read by the instrument. So it would not matter if the sample already has hemolyzed RBC from the long delay still should be the same reading. You can see that there is a positive bias running on the hemoglobin. So instead of disregarding this survey the site should investigation the bias in their hemoglobin.

EQA Example 4 RBC Problem
Here we have results on a CAP survey for a Pentra where the RBC and HCT have unacceptable results. For this instrument the Hct is calculated using the RBC and MCV. The last three samples have positive bias for RBC and Hct and the Hgb has negative bias.

So here we have the investigation results
So here we have the investigation results. I want you to note the area highlighted. It says that the QC looked good and comparison between the instruments was also fine. They also used manufacturer’s ranges. but said that the QC charts were all over the place corresponding to when service was made. So a good example that Internal QC can’t always let you know there are any problems. Also there doesn’t seem to be any policy for “out-of- control QC” They go on to say they that they replaced this instrument.

EQA Example 5 Multiple Analyte Problems
This is our last example was on a Abbott Again we have multiple failures. All the failures have positive bias.

On these 3 question on the investigation about the QC they said that the QC was out 2 SD
that there were biases but the QC was close to the manufacturer’s upper limit. First as we talked about in the internal QC section you need policy for “out of range” QC. Why didn’t the staff running samples that day question whether there was a problem with the instrument. They ran the survey even though the QC had multiple problems. It is very important that you staff understand how to look at the instrument QC and respond to correctly. Next we see the long corrective action. It appears that eventually they changed enough parts to correct the problem but still noticed they needed to calibrate. Who knows how long they would have continued to run patients on this instrument had the EQA showed major problems.

So we are coming to the end of our journey and the finish line can be seen.

Summary and Conclusion
Standardization Pre Analytic Control Post Analytic Control Analytical Control Internal Quality Control External Quality Assessment To wrap up my presentation I will now re-cap the 5 main points for necessary to undertake a program of Quality Management in Hematology Laboratory. They are Standardization – Involves the standardization of methods and the use of reference material standards to ensure correct resulting. As well as SOP that include troubleshooting. The creation of SOP help to maintain quality of performance by providing a stable pattern of function and avoiding unauthorized individual variation. Ensures consistent quality of work no matter what skill level from new to seasoned staff choosing the correct instrument/test for your needs. What are some of the Pre analytical components? - Knowing the function of your instrument. Knowing the start-up and shut-down procedure. Knowing through both sight and sound if you instrument is functioning correct. And of course the proper sample. If it was collected, stored, correctly. Then the Post analytical Control. – The resulting phase. Includes what? Know your instruments flags. Knowing when you need to check slides. Know if the results are humanly possible. Then we looked at the analytical phase that include internal Quality control I showed you several choice for the internal control. They were – commercial controls, retained patients and Moving average. It is better to use at least two of these if not all three. I am quite partial to the moving average since it can tell you within a day if you have instrument problems as long as your volume of samples is 100 or more. Then finally External Quality Assessment. Even if all you internal controls appear good, there is always a chance that there is still an instrument problem going undetected. Also can show you if your QC policies are being followed. It is always important to actually look at the report to see if you can see any biases, trends or shifts that do not correspond with your internal control.

Questions and Answers?

References Quality Control (QC) Information and Troubleshooting Guide – Hematology – Beckman Coulter The use of retained patient specimens for hematology quality control. Hackney JR, Cembrowski GS An optimized quality control procedure for hematology analyzers with the use of retained patient specimens. Cembrowski GS, Lunetzky ES, Patrick CC, Wilson MK Establishing Quality Control Means and Standard Deviation for Hematology Instrument, Streck Quality Assurance in Hematology, WHO

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