Using Root Cause Analysis

To illustrate the use of Root Cause Analysis, let us consider an example of troubleshooting where an IVF Lab Director is concerned about the poor quality of the sperm preparations that are being produced by the laboratory (see Table 7.5). The pertinent technical background on sperm preparation methods has been extensively reviewed previously (Mortimer, 2000; Mortimer and Mortimer, 1992) and will not be repeated here.

Issue Even with normal semen samples our sperm preparation method provides a low relative yield (about 15% of the motile spermatozoa initially applied to the gradient) with only 65-70% progressively motile spermatozoa and frequent contamination of the post-gradient sperm population with other cells and debris. A swim-up is needed to improve the motility, but that takes more time and further reduces the yield. Method The SOP for sperm preparation in our imaginary IVF lab can be summarized as follows. Apply 1 ml of liquefied semen to a gradient comprised of 0.5 ml layers of 80% and 40% PureSperm (Nidacon International AB, Goteborg, Sweden) in a Falcon 2003 tube. Spin at 2000 rpm for 15 minutes in a Centra CL2 centrifuge fitted with a model 809 rotor. Remove and discard the supernatant (seminal plasma and gradient layers) and resuspend

Table 7.5 An example of troubleshooting using RCA to address the issue of "Why do our sperm preparations have variable, and often low, proportions of progressively motile spermatozoa, and are contaminated by other cellular elements and debris from the original semen sample?"

Contributory factor





Gradient colloid concentrations

Gradient layer volumes

Centrifuge tube diameter

Centrifuge tube shape Fixed-angle centrifuge rotor

Centrifuge buckets are not sealed

Non-contributory Colloid concentrations of 40% and 80% are the recommended layers for PureSperm gradients.

Contributory Only 0.5 ml layers, this will lead to more rapid "raft" creation and blockage of sperm passage to the lower layer(s) -hence reducing the yield.

Contributory The tube diameter is relatively small, hence the layer interface area is reduced. This leads to more rapid "raft" creation and blockage of sperm passage to the lower layer(s) - hence reducing the yield.

Contributory A round bottom tube provides a less discrete pellet than a conical tube.

Contributory A fixed-angle rotor means that the pellet will not be deposited in the very bottom of the tube but "smeared" over a larger area of the bottom and one side.

Non-contributory Risk of aerosol contamination in the laboratory if a tube were to break during centrifugation.

None required.

Use larger volume layers of 1.5 or 1 2.0 ml.

Use a larger diameter tube, e.g. 1

Falcon 2095.

Use a conical bottom tube, e.g. 1

Falcon 2095. Change to a centrifuge that has a 1 swing-out or swinging bucket type rotor.

None in this regard.

Contributory factor





Centrifugation speed ofthe first spin

Centrifugation time of the first spin

Technique for harvesting the pellet from the gradient


Contributory Contributory

Resuspending the pellet in gradient tube

Centrifugation speed of the second spin



The stated rotor has a radius of 12.7 cm, giving a centrifugation force of approximately 570g which is higher than required.

Only 15 minutes might not be sufficient for the spermatozoa to reach their isopycnic points on the gradient.

Removal of all the layers above the pellet exposes the pellet to contamination by residual material from the upper layers that contaminates the inner surface of the tube.

The pellet will be contaminated with residual material from the seminal plasma and upper layers (and "rafts" of poor/dead sperm, other cells and debris) that coats the inner surface of the tube.

Although 570g is above the recommended 500 g, centrifugation force does not become harmful until 800 g.

Decrease the speed to give a centrifugation force of 300 g.

Increase to 20 minutes.

Remove only the seminal plasma layer, the upper (semen/40% colloid) "raft," 40% colloid layer and the 40/80 "raft" to leave most of the 80% colloid layer intact to protect the pellet from contamination. Then harvest the pellet by aspiration through the remaining 80% colloid layer.

After recovering the pellet from underneath the remainder of the 80% colloid layer, transfer it to a clean conical tube before resuspending the cells in fresh culture medium.

None required, but the centrifugation speed should be changed to 500 g so as to conform to the standard protocols.

Centrifugation time of the second spin Harvesting of the washed sperm pellet

Resuspension of the washed sperm pellet Performing a second wash step

Swim-up from the resuspended washed sperm suspension

Non-contributory Contributory

Non-contributory Contributory


15 minutes is the usual duration for this spin.

Because a round bottom tube was used in a fixed-angle rotor the pellet will be "smeared" over a larger area than if a conical tube had been used in a swing-out rotor, rendering complete harvesting more difficult.


Each wash cycle causes the loss of some spermatozoa, and this could contribute to the perceived low yield.

Although many labs perform such a procedure it is not necessary if the density gradient procedure is performed correctly The presence of even 15% immotile sperm in the final preparation has never been shown to be harmful to either the fertilization rate or embryo quality.

None required.

Change to a centrifuge that has a swing-out or swinging bucket type rotor and use a conical bottom tube, e.g. Falcon 2095.

None required.

Since this step is not necessary it should be omitted from the procedure. Do not perform this step.

1.As per manufacturer's instructions and established optimized methodology.

2.While this factor is non-contributory to the current issue, it does, however, represent a risk of possible aerosol contamination of the laboratory if a tube breaks while being centrifuged. In accordance with good laboratory practice the centrifuge should be upgraded to one with sealed buckets. In this case, this could be achieved by replacing only the rotor.

the pellet in 1 ml of fresh medium. Spin again at 2000 rpm for 15 minutes and again aspirate and discard the supernatant. Repeat the washing cycle. Resuspend the final sperm pellet in a small volume of fresh medium and overlay with 0.6 ml fresh medium then allow to swim up for 30-60 minutes at 37 °C in a CO2 incubator. Recover the upper 2/3 of the overlay and assess.

Constructing the RCA The process map for sperm preparation is shown in Figure 7.5. It clearly illustrates the need to delve deeper until no further subordinate level processes remain because, in reality, the sperm preparation process actually comprises three separate processes. Only by having dissected the process to this level can the following comprehensive troubleshooting exercise be undertaken. The Contributory Factors and their classifications are listed in Table 7.5. Benchmark criteria are taken from the recommended method for using PureSperm density gradients as per the manufacturer's package insert and previously published information.

Conclusions of the RCA While the method might not seem to be too different from that used by many other labs, there are numerous factors within the sperm preparation protocol that can contribute to reduced yield in quantitative and/or qualitative terms. In many cases the individual variation might not cause a

Figure 7.5 A process map for sperm preparation in IDEF0 format. The first panel shows the obligatory parent process map which is then broken down into sub-processes displayed in multiple, hierarchically linked process maps. Each map should have no more than 6 steps (although 9 could be used without breaking the formal numbering convention). The first map (second panel of Figure) shows the basic tasks involved in the process (steps 1 to 6), with step 2 being shown in greater detail in the third panel. The annotation "A2" outside the bottom right-hand corner of step 2 in the 6-step chart of process A-0 (its "node number") shows that the box has been detailed as a "child" diagram (i.e. the third panel of the Figure). In diagram A2, steps 1 and 2 are identified as being displayed in greater detail in child diagrams A21 and A22 (thefourth and fifth panels of the Figure). For clarity, information concerning the controls and mechanisms have been omitted.

marked degradation in the outcome, but taken together there will be substantial detrimental impact. The final conclusion is that the sperm preparation method should be replaced by one based on the manufacturer's instructions and established optimized methodology. Furthermore, because this change is not going to have an unknown or uncertain outcome on the process, there is no need to perform any validation studies of the "new" method.

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100 Pregnancy Tips

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